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Macrophages are key players of the innate immune system that can roughly be divided into the pro-inflammatory M1 type and the anti-inflammatory, pro-healing M2 type. While a transient initial pro-inflammatory state is helpful, a prolonged inflammation deteriorates a proper healing and subsequent regeneration. One promising strategy to drive macrophage polarization by biomaterials is precise control over biomaterial geometry. For regenerative approaches, it is of particular interest to identify geometrical parameters that direct human macrophage polarization. For this purpose, we advanced melt electrowriting (MEW) towards the fabrication of fibrous scaffolds with box-shaped pores and precise inter-fiber spacing from 100 μm down to only 40 μm. These scaffolds facilitate primary human macrophage elongation accompanied by differentiation towards the M2 type, which was most pronounced for the smallest pore size of 40 μm. These new findings can be important in helping to design new biomaterials with an enhanced positive impact on tissue regeneration.
In recent decades, hybrid characterization systems have become pillars in the study of cellular biomechanics. Especially, Atomic Force Microscopy (AFM) is combined with a variety of optical microscopy techniques to discover new aspects of cell adhesion. AFM, however, is limited to the early-stage of cell adhesion, so that the forces of mature cell contacts cannot be addressed. Even though the invention of Fluidic Force Microscopy (FluidFM) overcomes these limitations by combining the precise force-control of AFM with microfluidics, the correlative investigation of detachment forces arising from spread mammalian cells has been barely achieved. Here, a novel multifunctional device integrating Fluorescence Microscopy (FL) into FluidFM technology (FL-FluidFM) is introduced, enabling real-time optical tracking of entire cell detachment processes in parallel to the undisturbed acquisition of force-distance curves. This setup, thus, allows for entailing two pieces of information at once. As proof-of-principle experiment, this method is applied to fluorescently labeled rat embryonic fibroblast (REF52) cells, demonstrating a precise matching between identified force-jumps and visualized cellular unbinding steps. This study, thus, presents a novel characterization tool for the correlated evaluation of mature cell adhesion, which has great relevance, for instance, in the development of biomaterials or the fight against diseases such as cancer.
Biointerface engineering is a wide-spread strategy to improve the healing process and subsequent tissue integration of biomaterials. Especially the integration of specific peptides is one promising strategy to promote the regenerative capacity of implants and 3D scaffolds. In vivo, these tailored interfaces are, however, first confronted with the innate immune response. Neutrophils are cells with pronounced proteolytic potential and the first recruited immune cells at the implant site; nonetheless, they have so far been underappreciated in the design of biomaterial interfaces. Herein, an in vitro approach is introduced to model and analyze the neutrophil interaction with bioactivated materials at the example of nano-bioinspired electrospun surfaces that reveals the vulnerability of a given biointerface design to the contact with neutrophils. A sacrificial, transient hydrogel coating that demonstrates optimal protection for peptide-modified surfaces and thus alleviates the immediate cleavage by neutrophil elastase is further introduced.
Supplement-free induction of cellular differentiation and polarization solely through the topography of materials is an auspicious strategy but has so far significantly lagged behind the efficiency and intensity of media-supplementation-based protocols. Consistent with the idea that 3D structural motifs in the extracellular matrix possess immunomodulatory capacity as part of the natural healing process, it is found in this study that human-monocyte-derived macrophages show a strong M2a-like prohealing polarization when cultured on type I rat-tail collagen fibers but not on collagen I films. Therefore, it is hypothesized that highly aligned nanofibrils also of synthetic polymers, if packed into larger bundles in 3D topographical biomimetic similarity to native collagen I, would induce a localized macrophage polarization. For the automated fabrication of such bundles in a 3D printing manner, the strategy of “melt electrofibrillation” is pioneered by the integration of flow-directed polymer phase separation into melt electrowriting and subsequent selective dissolution of the matrix polymer postprocessing. This process yields nanofiber bundles with a remarkable structural similarity to native collagen I fibers, particularly for medical-grade poly(ε-caprolactone). These biomimetic fibrillar structures indeed induce a pronounced elongation of human-monocyte-derived macrophages and unprecedentedly trigger their M2-like polarization similar in efficacy as interleukin-4 treatment.
Abstract
Ligaments and tendons are comprised of aligned, crimped collagen fibrils that provide tissue-specific mechanical properties with non-linear extension behaviour, exhibiting low stress at initial strain (toe region behaviour). To approximate this behaviour, we report fibrous scaffolds with sinusoidal patterns by melt electrowriting (MEW) below the critical translation speed (CTS) by exploitation of the natural flow behaviour of the polymer melt. More specifically, we synthesised photopolymerizable poly(L-lactide-co-ε-caprolactone-co-acryloyl carbonate) (p(LLA-co-ε-CL-co-AC)) and poly(ε-caprolactone-co-acryloyl carbonate) (p(ε-CL-co-AC)) by ring-opening polymerization (ROP). Single fibre (fØ = 26.8 ± 1.9 µm) tensile testing revealed a customisable toe region with Young’s Moduli ranging from E = 29 ± 17 MPa for the most crimped structures to E = 314 ± 157 MPa for straight fibres. This toe region extended to scaffolds containing multiple fibres, while the sinusoidal pattern could be influenced by printing speed. The synthesized polymers were cytocompatible and exhibited a tensile strength of σ = 26 ± 7 MPa after 104 cycles of preloading at 10% strain while retaining the distinct toe region commonly observed in native ligaments and tendon tissue.
Statement of Significance
Damaged tendons and ligaments are serious and frequently occurring injuries worldwide. Recent therapies, including autologous grafts, still have severe disadvantages leading to a demand for synthetic alternatives. Materials envisioned to induce tendon and ligament regeneration should be degradable, cytocompatible and mimic the ultrastructural and mechanical properties of the native tissue. Specifically, we utilised photo-cross-linkable polymers for additive manufacturing (AM) with MEW. In this way, we were able to direct-write cytocompatible fibres of a few micrometres thickness into crimp-structured elastomer scaffolds that mimic the non-linear biomechanical behaviour of tendon and ligament tissue.
Melt electrowriting (MEW) is an additive manufacturing technology that is recently used to fabricate voluminous scaffolds for biomedical applications. In this study, MEW is adapted for the seeding of multicellular spheroids, which permits the easy handling as a single sheet-like tissue-scaffold construct. Spheroids are made from adipose-derived stromal cells (ASCs). Poly(ε-caprolactone) is processed via MEW into scaffolds with box-structured pores, readily tailorable to spheroid size, using 13–15 µm diameter fibers. Two 7–8 µm diameter “catching fibers” near the bottom of the scaffold are threaded through each pore (360 and 380 µm) to prevent loss of spheroids during seeding. Cell viability remains high during the two week culture period, while the differentiation of ASCs into the adipogenic lineage is induced. Subsequent sectioning and staining of the spheroid-scaffold construct can be readily performed and accumulated lipid droplets are observed, while upregulation of molecular markers associated with successful differentiation is demonstrated. Tailoring MEW scaffolds with pores allows the simultaneous seeding of high numbers of spheroids at a time into a construct that can be handled in culture and may be readily transferred to other sites for use as implants or tissue models.
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.