A Spatiotemporal Controllable Biomimetic Skin for Accelerating Wound Repair.

Skin injury repair is a dynamic process involving a series of interactions over time and space. Linking human physiological processes with materials' changes poses a significant challenge. To match the wound healing process, a spatiotemporal controllable biomimetic skin is developed, which comprises a three-dimensional (3D) printed membrane as the epidermis, a cell-containing hydrogel as the dermis, and a cytokine-laden hydrogel as the hypodermis. In the initial stage of the biomimetic skin repair wound, the membrane frame aids wound closure through pre-tension, while cells proliferate within the hydrogel. Next, as the frame disintegrates over time, cells released from the hydrogel migrate along the residual membrane. Throughout the process, continuous cytokines release from the hypodermis hydrogel ensures comprehensive nourishment. The findings reveal that in the rat full-thickness skin defect model, the biomimetic skin demonstrated a wound closure rate eight times higher than the blank group, and double the collagen content, particularly in the early repair process. Consequently, it is reasonable to infer that this biomimetic skin holds promising potential to accelerate wound closure and repair. This biomimetic skin with mechanobiological effects and spatiotemporal regulation emerges as a promising option for tissue regeneration engineering.

Melt Electrowriting of Nylon-12 Microfibers with an Open-Source 3D Printer.

This study demonstrates how either a heated flat or cylindrical collector enables defect-free melt electrowriting (MEW) of complex geometries from high melting temperature polymers. The open-source "MEWron" printer uses nylon-12 filament and combined with a heated flat or cylindrical collector, produces well-defined fibers with diameters ranging from 33 ± 4 to 95 ± 3 µm. Processing parameters for stable jet formation and minimal defects based on COMSOL thermal modeling for hardware design are optimized. The balance of processing temperature and collector temperature is achieved to achieve auxetic patterns, while showing that annealing nylon-12 tubes significantly alters their mechanical properties. The samples exhibit varied pore sizes and wall thicknesses influenced by jet dynamics and fiber bridging. Tensile testing shows nylon-12 tubes are notably stronger than poly(ε-caprolactone) ones and while annealing has limited impact on tensile strength, yield, and elastic modulus, it dramatically reduces elongation. The equipment described and material used broadens MEW applications for high melting point polymers and highlights the importance of cooling dynamics for reproducible samples.

The Multiweek Thermal Stability of Medical-Grade Poly(ε-caprolactone) During Melt Electrowriting.

Melt electrowriting (MEW) is a high-resolution additive manufacturing technology that places unique constraints on the processing of thermally degradable polymers. With a single nozzle, MEW operates at low throughput and in this study, medical-grade poly(ε-caprolactone) (PCL) is heated for 25 d at three different temperatures (75, 85, and 95 °C), collecting daily samples. There is an initial increase in the fiber diameter and decrease in the jet speed over the first 5 d, then the MEW process remains stable for the 75 and 85 °C groups. When the collector speed is fixed to a value at least 10% above the jet speed, the diameter remains constant for 25 d at 75 °C and only increases with time for 85 and 95 °C. Fiber fusion at increased layer height is observed for 85 and 95 °C, while the surface morphology of single fibers remain similar for all temperatures. The properties of the prints are assessed with no observable changes in the degree of crystallinity or the Young's modulus, while the yield strength decreases in later phases only for 95 °C. After the initial 5-d period, the MEW processing of PCL at 75 °C is extraordinarily stable with overall fiber diameters averaging 13.5 ± 1.0 µm over the entire 25-d period.

Medical-grade polycaprolactone scaffolds made by melt electrospinning writing for oral bone regeneration - a pilot study in vitro.

BACKGROUND: The spectrum of indications for the use of membranes and scaffolds in the field of oral and maxillofacial surgery includes, amongst others, guided bone regeneration (GBR). Currently available membrane systems face certain disadvantages such as difficult clinical handling, inconsistent degradation, undirected cell growth and a lack of stability that often complicate their application. Therefore, new membranes which can overcome these issues are of great interest in this field. METHODS: In this pilot study, we investigated polycaprolactone (PCL) scaffolds intended to enhance oral wound healing by means of melt electrospinning writing (MEW), which allowed for three-dimensional (3D) printing of micron scale fibers and very exact fiber placement. A singular set of box-shaped scaffolds of different sizes consisting of medical-grade PCL was examined and the scaffolds' morphology was evaluated via scanning electron microscopy (SEM). Each prototype sample with box sizes of 225 µm, 300 µm, 375 µm, 450 µm and 500 µm was assessed for cytotoxicity and cell growth by seeding each scaffold with human osteoblast-like cell line MG63. RESULTS: All scaffolds demonstrated good cytocompatibility according to cell viability, protein concentration, and cell number. SEM analysis revealed an exact fiber placement of the MEW scaffolds and the growth of viable MG63 cells on them. For the examined box-shaped scaffolds with pore sizes between 225 µm and 500 µm, a preferred box size for initial osteoblast attachment could not be found. CONCLUSIONS: These well-defined 3D scaffolds consisting of medical-grade materials optimized for cell attachment and cell growth hold the key to a promising new approach in GBR in oral and maxillofacial surgery.

Development of Endothelial Cell Networks in 3D Tissues by Combination of Melt Electrospinning Writing with Cell-Accumulation Technology.

A remaining challenge in tissue engineering approaches is the in vitro vascularization of engineered constructs or tissues. Current approaches in engineered vascularized constructs are often limited in the control of initial vascular network geometry, which is crucial to ensure full functionality of these constructs with regard to cell survival, metabolic activity, and potential differentiation ability. Herein, the combination of 3D-printed poly-ε-caprolactone scaffolds via melt electrospinning writing with the cell-accumulation technique to enable the formation and control of capillary-like network structures is reported. The cell-accumulation technique is already proven itself to be a powerful tool in obtaining thick (50 µm) tissues and its main advantage is the rapid production of tissues and its ease of performance. However, the applied combination yields tissue thicknesses that are doubled, which is of outstanding importance for an improved handling of the scaffolds and the generation of clinically relevant sample volumes. Moreover, a correlation of increasing vascular endothelial growth factor secretion to hypoxic conditions with increasing pore sizes and an assessment of the formation of neovascular like structures are included.

Dimension-Based Design of Melt Electrowritten Scaffolds.

The electrohydrodynamic stabilization of direct-written fluid jets is explored to design and manufacture tissue engineering scaffolds based on their desired fiber dimensions. It is demonstrated that melt electrowriting can fabricate a full spectrum of various fibers with discrete diameters (2-50 µm) using a single nozzle. This change in fiber diameter is digitally controlled by combining the mass flow rate to the nozzle with collector speed variations without changing the applied voltage. The greatest spectrum of fiber diameters was achieved by the simultaneous alteration of those parameters during printing. The highest placement accuracy could be achieved when maintaining the collector speed slightly above the critical translation speed. This permits the fabrication of medical-grade poly(ε-caprolactone) into complex multimodal and multiphasic scaffolds, using a single nozzle in a single print. This ability to control fiber diameter during printing opens new design opportunities for accurate scaffold fabrication for biomedical applications.

Melt Electrowriting of Thermoplastic Elastomers.

Melt electrowriting (MEW), an additive manufacturing process, is established using polycaprolactone as the benchmark material. In this study, a thermoplastic elastomer, namely, poly(urea-siloxane), is synthesized and characterized to identify how different classes of polymers are compatible with MEW. This polyaddition polymer has reversible hydrogen bonding from the melt upon heating/cooling and highly resolved structures are achieved by MEW. The influence of applied voltage, temperature, and feeding pressure on printing outcomes behavior is optimized. Balancing these parameters, highly uniform and smooth-surfaced fibers with diameters ranging from 10 to 20 µm result. The quality of the 3D MEW scaffolds is excellent, with very accurate fiber stacking capacity-up to 50 layers with minimal defects and good fiber fusion between the layers. There is also minimal fiber sagging between the crossover points, which is a characteristic of thicker MEW scaffolds previously reported with other polymers. In summary, poly(urea-siloxane) demonstrates outstanding compatibility with the MEW process and represents a class of polymer-thermoplastic elastomers-that are, until now, untested with this approach.

Microfibrous Scaffolds Guide Stem Cell Lumenogenesis and Brain Organoid Engineering.

3D organoids are widely used as tractable in vitro models capable of elucidating aspects of human development and disease. However, the manual and low-throughput culture methods, coupled with a low reproducibility and geometric heterogeneity, restrict the scope and application of organoid research. Combining expertise from stem cell biology and bioengineering offers a promising approach to address some of these limitations. Here, melt electrospinning writing is used to generate tuneable grid scaffolds that can guide the self-organization of pluripotent stem cells into patterned arrays of embryoid bodies. Grid geometry is shown to be a key determinant of stem cell self-organization, guiding the position and size of emerging lumens via curvature-controlled tissue growth. Two distinct methods for culturing scaffold-grown embryoid bodies into either interconnected or spatially discrete cerebral organoids are reported. These scaffolds provide a high-throughput method to generate, culture, and analyze large numbers of organoids, substantially reducing the time investment and manual labor involved in conventional methods of organoid culture. It is anticipated that this methodological development will open up new opportunities for guiding pluripotent stem cell culture, studying lumenogenesis, and generating large numbers of uniform organoids for high-throughput screening.

Effects of Six Processing Parameters on the Size of PCL Fibers Prepared by Melt Electrospinning Writing.

Melt electrospinning writing is a new and promising method for fabricating micro/nanofibers, which has shown great prospects in the biomedical fields such as 3D printing of porous scaffolds. The diameter of the melt electrospinning writing fiber can determine the resolution of the microstructure; thus, the controllability of the fiber diameter is of great significance to the whole fabrication process. In this paper, an orthogonal design experiment (six factors, three levels) was used to explore the impacts of six melt electrospinning parameters (melt temperature, collector speed, tip-to-collector distance, melt flow rate, voltage, and needle gauge) on the fiber diameter. In this experiment, the diameter of fibers obtained with the designed experimental parameters and conditions varied from 10.30 µm to 20.02 µm. The range analysis of orthogonal test results showed that the melt flow rate was the most important factor influencing the diameter of melt electrospinning writing fiber, while the voltage was the least influential factor. The variance analysis of orthogonal test results showed that melt temperature, collector velocity, tip-to-collector distance and melt flow rate had a significant influence on the diameter of melt electrospinning writing fiber. On the basis of the first-order regression equation, the fiber diameter of poly-ε-caprolactone can be accurately controlled, thus improving the engineering applications of poly-ε-caprolactone.

A Decade of Melt Electrowriting.

Over the past decade, melt electrowriting (MEW) has established the fundamental understanding of processing (and printer) requirements. Iterative work on parametric development and dissemination of this recent additive manufacturing technology has been performed across many systems and polymers (mainly poly-(ε-caprolactone)), showing similarities and trends. However, the software and hardware ecosystems of MEW are not mature. Further, due to its multi-parametric nature, MEW can be challenging for laboratories to master. This review intends to provide a unique perspective on the dynamic relationship between MEW processing parameters. Such parameters can be divided into 1) those that affect the polymer flow rate to or 2) from the nozzle, and 3) environmental conditions. The most influential parameters for high-quality printing are applied voltage, applied pressure, collector speed, polymer temperature, nozzle diameter, and the conditions that lead to charge buildup (e.g., relative humidity). Other factors such as ambient temperature, nozzle size, and protrusion, collector temperature and conductivity, and collector distance can all affect the process. Success for MEW printing means fibers fall onto the collector according to their pre-programmed path with predicted fiber diameter. Here, the authors elucidate how the dynamic relationship between these parameters can converge into ideal printing conditions to produce scaffolds.

Cell Seeding on 3D Scaffolds for Tissue Engineering and Disease Modeling Applications.

Scaffold cell seeding is a crucial step for the standardization and homogeneous maturation of tissue engineered constructs. This is particularly critical in the context of additively manufactured scaffolds whereby large pore size and high porosity usually impedes the retention of the seeding solution resulting in poor seeding efficacy and heterogeneous cell distribution. To circumvent this limitation, a simple yet efficient cell seeding technique is described in this chapter consisting of preincubating the scaffold in 100% serum for 1 h leading to reproducible seeding. A proof of concept is demonstrated using highly porous melt electrowritten polycaprolactone scaffolds as the cell carrier. As cell density, cell distribution, and differentiation within the scaffold are important parameters, various assays are proposed to validate the seeding and perform quality control of the cellularized construct using techniques such as alizarin red, Sirius red, and immunostaining.

Fabrication of bioinspired grid-crimp micropatterns by melt electrospinning writing for bone-ligament interface study.

Many strategies have been adopted to engineer bone-ligament interface, which is of great value to both the tissue regeneration and the mechanism understanding underlying interface regeneration. However, how to recapitulate the complexity and heterogeneity of the native bone-ligament interface including the structural, cellular and mechanical gradients is still challenging. In this work, a bioinspired grid-crimp micropattern fabricated by melt electrospinning writing (MEW) was proposed to mimic the native structure of bone-ligament interface. The printing strategy of crimped fiber micropattern was developed and the processing parameters were optimized, which were used to mimic the crimp structure of the collagen fibrils in ligament. The guidance effect of the crimp angle and fiber spacing on the orientation of fibroblasts was studied, and both of them showed different levels of cell alignment effect. MEW grid micropatterns with different fiber spacings were fabricated as bone region. Both the alkaline phosphatase activity and calcium mineralization results demonstrated the higher osteoinductive ability of the MEW grid structures, especially for that with smaller fiber spacing. The combined grid-crimp micropatterns were applied for the co-culture of fibroblasts and osteoblasts. The results showed that more cells were observed to migrate into the in-between interface region for the pattern with smaller fiber spacing, suggested the faster migration speed of cells. Finally, a cylindrical triphasic scaffold was successfully generated by rolling the grid-crimp micropatterns up, showing both structural and mechanical similarity to the native bone-ligament interface. In summary, the proposed strategy is reliable to fabricate grid-crimp triphasic micropatterns with controllable structural parameters to mimic the native bone-to-ligament structure, and the generated 3D scaffold shows great potential for the further bone-ligament interface tissue engineering.

Melt Electrospinning Writing of Mesh Implants for Pelvic Organ Prolapse Repair.

Over the past decade, melt electrospinning writing has attracted renewed attention. When combined with three-dimensional (3D) printing capabilities, complex 3D structures can be produced, from ultrafine fibers in the absence of toxic solvents, making it particularly attractive to fabricate customized scaffolds and implants for medical applications. This research aimed to develop novel less stiff vaginal mesh implants for pelvic organ prolapse (POP) repair, matching the physiological biomechanics of vaginal tissues. The main objectives, to attain that goal, were: development of a melt electrospinning writing prototype, with additive manufacturing capability, to produce complex structures from micrometer scale fibers, in a direct 3D printing mode; and design and validate new concepts of biodegradable meshes/scaffolds with new geometries, for POP repair. The melt electrospinning writing prototype was built based on different modules. Biodegradable polycaprolactone was used to produce novel implants: three geometries and two fiber configurations were employed. The commercially available Restorelle® (Coloplast) mesh was used as a benchmark. Printed implants were analyzed via scanning electron microscopy (SEM) and uniaxial tensile testing. The SEM images showed that the geometry is generally well produced; however, some minor deviations are visible due to charge interactions. The tensile test results indicated that, regardless of the geometry, the samples showed an elastic behavior for smaller displacements; aplastic behavior dominates later stages. In the physiological range of deformation, the novel meshes (80 µm fiber diameter) matched the tissue properties (p > 0.05). The Restorelle mesh was significantly stiffer than vaginal tissue (p < 0.05) and novel meshes. The precision of the various geometrical patterns and fiber diameters produced highlights the success of the designed and built prototype equipment. Results showed that the biodegradable meshes produced are biomechanically more compatible with native tissue than commercial implants.

Melt Electrospinning Writing of Magnetic Microrobots.

The magnetic microrobots actuated by an external magnetic field can access distant, enclosed, and small spaces under fuel-free conditions, which is apromising technology for manipulation and delivery under microenvironment; however, the complicated fabrication method limits their applications. Herein, three techniques including melt electrospinning writing (MEW), micromolding, and skiving process are combined to successfully mass-produce tadpole-like magnetic polycaprolactone/Fe3O4 (PCL/Fe3O4) microrobot. Importantly, the tadpole-like microrobots under an external magnetic field can achieve two locomotions: rolling mode and propulsion mode. The rolling motion can approach the working destination quickly with a speed of ≈2 mm s-1. The propulsion motion (0-340 µm s-1) can handle a microcargo. Such a simple and cost-effective production method shows a great potential for scale-up fabrication of advanced shape-design, mass-production, and multifunctionality microrobot.

Production of Scaffolds Using Melt Electrospinning Writing and Cell Seeding.

Melt electrospinning writing (MEW) is a solvent-free fabrication method for making polymer fiber scaffolds with features which include large surface area, high porosity, and controlled deposition of the fibers. These scaffolds are ideal for tissue engineering applications. Here we describe how to produce scaffolds made from poly(ε-caprolactone) using MEW and the seeding of primary human-derived dermal fibroblasts to create cell-scaffold constructs. The same methodology could be used with any number of cell types and MEW scaffold designs.

Convergence of Machine Vision and Melt Electrowriting.

Melt electrowriting (MEW) is a high-resolution additive manufacturing technology that balances multiple parametric variables to arrive at a stable fabrication process. The better understanding of this balance is underscored here using high-resolution camera vision of jet stability profiles in different electrical fields. Complementing this visual information are fiber-diameter measurements obtained at precise points, allowing the correlation to electrified jet properties. Two process signatures-the jet angle and for the first time, the Taylor cone area-are monitored and analyzed with a machine vision system, while SEM imaging for diameter measurement correlates real-time information. This information, in turn, allows the detection and correction of fiber pulsing for accurate jet placement on the collector, and the in-process assessment of the fiber diameter. Improved process control is used to successfully fabricate collapsible MEW tubes; structures that require exceptional accuracy and printing stability. Using a precise winding angle of 60° and 300 layers, the resulting 12 mm-thick tubular structures have elastic snap-through instabilities associated with mechanical metamaterials. This study provides a detailed analysis of the fiber pulsing occurrence in MEW and highlights the importance of real-time monitoring of the Taylor cone volume to better understand, control, and predict printing instabilities.

Polymers for Melt Electrowriting.

Melt electrowriting (MEW) is an emerging high-resolution additive manufacturing technique based on the electrohydrodynamic processing of polymers. MEW is predominantly used to fabricate scaffolds for biomedical applications, where the microscale fiber positioning has substantial implications in its macroscopic mechanical properties. This review gives an update on the increasing number of polymers processed via MEW and different commercial sources of the gold standard poly(ε-caprolactone) (PCL). A description of MEW-processed polymers beyond PCL is introduced, including blends and coated fibers to provide specific advantages in biomedical applications. Furthermore, a perspective on printer designs and developments is highlighted, to keep expanding the variety of processable polymers for MEW.

Effect of Dual Pore Size Architecture on In Vitro Osteogenic Differentiation in Additively Manufactured Hierarchical Scaffolds.

The combination of macro- and microporosity is a potent manner of enhancing osteogenic potential, but the biological events leading to this increase in osteogenesis are not well understood. In this study, we investigated the effect of a dual pore size scaffold on the physical and biological properties, with the hypothesis that cell condensation is the determining factor for enhanced osteogenic differentiation. To this end, a hierarchical scaffold possessing a dual (large and small) pore size was fabricated by combining two additive manufacturing techniques: melt electrospinning writing (MEW) and fused deposition modeling (FDM). The scaffolds showed a mechanical stiffness of 23.2 ± 1.5 MPa similar to the FDM control scaffold, while the hybrid revealed an increased specific surface area of 1.4 ± 0.1 m2/g. The scaffold was cultured with primary human osteoblasts for 28 days, which showed enhanced cell adhesion and proliferation. The hierarchical structure was also beneficial for in vitro alkaline phosphate activity and mineralization and showed an increased expression of osteogenic protein and genes. Mesenchymal condensation markers related to osteoblastic differentiation (CDH2, RhoA, Rac1, and Cdc42) were upregulated in the hybrid construct, demonstrating that the MEW membrane provided an environment more suitable for the recapitulation of cell condensation, which in turn leads to higher osteogenic differentiation. In summary, this study demonstrated that the hierarchical scaffold developed in this paper leads to a significant improvement in the scaffold properties such as increased specific surface area, initial cell adhesion, cell proliferation, and in vitro osteogenesis.

Highly Ordered 3D Tissue Engineering Scaffolds as a Versatile Culture Platform for Nerve Cells Growth.

Tissue engineering scaffolds provide an encouraging alternative for nerve injuries due to their biological support for nerve cell growth, which can be used for neuronal repair. Nerve cells have been reported to be mostly cultured on 2D scaffolds that cannot mimic the native extracellular matrix. Herein, highly ordered 3D scaffolds are fabricated for nerve cell culture by melt electrospinning writing, the microstructures and geometries of the scaffolds could be well modulated. An effective strategy for scaffold surface modification to promote nerve cell growth is proposed. The effects of scaffolds with different surface modifications, viz., plasma treatment, single poly-D-lysine (PDL) coating after plasma treatment, single laminin (LM) coating after plasma treatment, double PDL and LM coatings after plasma treatment, on PC12 cell growth are evaluated. Experiments show the scaffold modified with double PDL and LM coatings after plasma treatment facilitated the growth of PC12 cells most effectively, indicating the synergistic effect of PDL and LM on the growth of nerve cells. This is the first systematic and quantitative study of the effects of different scaffold surface modifications on nerve cell growth. The above results provide a versatile culture platform for growing nerve cells, and for recovery from peripheral nerve injury.

Fabrication and in vitro evaluation of PCL/gelatin hierarchical scaffolds based on melt electrospinning writing and solution electrospinning for bone regeneration.

As an emerging 3D printing technique, melt electrospinning writing (MEW) has been used to fabricate scaffolds with controllable structure and good mechanical strength for bone regeneration. However, how to further improve MEW scaffolds with nanoscale extracellular matrix (ECM) mimic structure and bioactivity is still challenging. In this study, we proposed a simple composite process by combining MEW and solution electrospinning (SE) to fabricate a micro/nano hierarchical scaffold for bone tissue engineering. The morphological results confirmed the hierarchical structure with both well-defined MEW microfibrous grid structure and SE random nanofiber morphology. The addition of gelatin nanofibers turned the scaffolds to be hydrophilic, and led to a slight enhancement of mechanical strength. Compared with PCL MEW scaffolds, higher cell adhesion efficiency, improved cell proliferation and higher osteoinductive ability were achieved for the MEW/SE composite scaffolds. Finally, multilayer composite scaffolds were fabricated by alternately stacking of MEW layer and SE layer and used to assess the effect on cell ingrowth in the scaffolds. The results showed that gelatin nanofibers did not inhibit cell penetration, but promoted the three-dimensional growth of bone cells. Thus, the strategy of the combined use of MEW and SE is a potential method to fabricate micro/nano hierarchical scaffolds to improve bone regeneration.