Search Results (15)

Three-dimensional (3D) scaffold systems have proven instrumental in advancing our understanding of polymicrobial biofilm dynamics and probiotic interactions within the oral environment. Among oral probiotics, Streptococcus salivarius K12 (Ssk12) has shown considerable promise in modulating microbial homeostasis; however, its long-term therapeutic benefits are contingent upon successful and sustained colonization of the oral mucosa. Despite its clinical relevance, the molecular mechanisms underlying the adhesion, persistence, and integration of Ssk12 into the native oral microbiome/biofilm remain inadequately characterized. In this pilot study, we explored the temporal colonization dynamics of Ssk12 and its impact on the structure and functional profiles of salivary-derived biofilms cultivated on melt-electrowritten poly(ε-caprolactone) (MEW-mPCL) scaffolds, which emulate the native oral niche. Colonization was monitored via fluorescence in situ hybridization (smFISH), confocal microscopy, and RT-qPCR, while shifts in community composition and function were assessed using 16S rRNA sequencing and meta-transcriptomics. A single administration of Ssk12 exhibited transient colonization lasting up to 7 days, with detectable presence diminishing by day 10. This was accompanied by short-term increases in Lactobacillus and Bifidobacterium populations. Functional analyses revealed increased transcriptional signatures linked to oxidative stress resistance and metabolic adaptation. These findings suggest that even short-term probiotic colonization induces significant functional changes, underscoring the need for strategies to enhance probiotic persistence. Full article

Musculoskeletal tissue injuries of the bone, cartilage, ligaments, tendons, and skeletal muscles are among the most common injuries experienced in medicine and become increasingly problematic in cases of significant tissue damage, such as nonunion bone defects and volumetric muscle loss. Current gold standard treatment options for musculoskeletal injuries, although effective, have limited capability to fully restore native tissue structure and function. To overcome this challenge, three-dimensional (3D) printing techniques have emerged as promising therapeutic options for tissue regeneration. Melt electrowriting (MEW), a recently developed advanced 3D printing technique, has gained significant traction in the field of tissue regeneration because of its ability to fabricate complex customizable scaffolds via high-precision microfiber deposition. The tailorability at microscale levels offered by MEW allows for enhanced recapitulation of the tissue microenvironment. Here, we survey the recent contributions of MEW in advancing musculoskeletal tissue engineering. More specifically, we briefly discuss the principles and technical aspects of MEW, provide an overview of current printers on the market, review in-depth the latest biomedical applications in musculoskeletal tissue regeneration, and, lastly, examine the limitations of MEW and offer future perspectives. Full article

The human trabecular meshwork (HTM) is responsible for regulating intraocular pressure (IOP) by means of gradient porosity. Changes in its physical properties, like increases in stiffness or alterations in the extracellular matrix (ECM), are associated with increases in the IOP, which is the primary cause of glaucoma. The complexity of its structure limits the engineered models to one-layered and simple approaches, which do not accurately replicate the biological and physiological cues related to glaucoma. Here, a combination of melt electrowriting (MEW) and solution electrospinning (SE) is explored as a biofabrication technique used to produce a gradient porous scaffold that mimics the multi-layered structure of the native HTM. Polycaprolactone (PCL) constructs with a height of 20–710 µm and fiber diameters of 0.7–37.5 µm were fabricated. After mechanical characterization, primary human trabecular meshwork cells (HTMCs) were seeded over the scaffolds within the subsequent 14–21 days. In order to validate the system’s responsiveness, cells were treated with dexamethasone (Dex) and the rho inhibitor Netarsudil (Net). Scanning electron microscopy and immunochemistry staining were performed to evaluate the expected morphological changes caused by the drugs. Cells in the engineered membranes exhibited an HTMC-like morphology and a correct drug response. Although this work demonstrates the utility of combining MEW and SE in reconstructing complex morphological features like the HTM, new geometries and dimensions should be tested, and future works need to be directed towards perfusion studies. Full article

Medical device-related infections (DRIs), especially prevalent among critically ill patients, impose significant health and economic burdens and are mainly caused by bacteria. Severe infections often necessitate device removal when antibiotic therapy is inefficient, delaying recovery. To tackle this issue, PCL drug-eluting coated meshes were explored, and they were printed via melt electrowriting (MEW). These meshes were coated with gentamicin sulfate (GS) and tetracycline hydrochloride (TCH) and underwent FTIR analysis to confirm drug integration. Antimicrobial activity was assessed via agar diffusion assays and biofilm formation assays against bacterial strains: Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 43300, and Staphylococcus epidermidis ATCC 35984. FTIR analysis evidenced the presence of the drugs in the meshes. TCH displayed broad-spectrum antimicrobial activity against all strains, whereas GS was effective against all except S. aureus. These findings indicate the potential of cost-effective ultra-fine drug coating fibers for medical device applications, offering infection prevention during implantation. This preliminary study demonstrates the feasibility of producing drug-eluting fibers for DRI prevention through a non-toxic, fast, and cost-efficient technique, paving the way for enhanced patient care and reduced healthcare costs. Full article

As microfiber-based additive manufacturing (AM) technologies, melt electrowriting (MEW) and solution electrowriting (SEW) have demonstrated efficacy with more biomedically relevant materials. By processing SU-8 resin using MEW and SEW techniques, a material with substantially different mechanical, thermal, and optical properties than that typically processed is introduced. SU-8 polymer is temperature sensitive and requires the devising of a specific heating protocol to be properly processed. Smooth-surfaced microfibers resulted from MEW of SU8 for a short period (from 30 to 90 min), which provides the greatest control and, thus, reproducibility of the printed microfibers. This investigation explores various parameters influencing the electrowriting process, printing conditions, and post-processing to optimize the fabrication of intricate 3D structures. This work demonstrates the controlled generation of straight filaments and complex multi-layered architectures, which were characterized by brightfield, darkfield, and scanning electron microscopy (SEM). This research opens new avenues for the design and development of 3D-printed photonic systems by leveraging the properties of SU-8 after both MEW and SEW processing. Full article

We demonstrate for the first time the combination of two additive manufacturing technologies used in tandem, fused deposition modelling (FDM) and melt electrowriting (MEW), to increase the range of possible MEW structures, with a focus on creating branched, hollow scaffolds for vascularization. First, computer-aided design (CAD) was used to design branched mold halves which were then used to FDM print conductive polylactic acid (cPLA) molds. Next, MEW was performed over the top of these FDM cPLA molds using polycaprolactone (PCL), an FDA-approved biomaterial. After the removal of the newly constructed MEW scaffolds from the FDM molds, complementary MEW scaffold halves were heat-melded together by placing the flat surfaces of each half onto a temperature-controlled platform, then pressing the heated halves together, and finally allowing them to cool to create branched, hollow constructs. This hybrid technique permitted the direct fabrication of hollow MEW structures that would otherwise not be possible to achieve using MEW alone. The scaffolds then underwent in vitro physical and biological testing. Specifically, dynamic mechanical analysis showed the scaffolds had an anisotropic stiffness of 1 MPa or 5 MPa, depending on the direction of the applied stress. After a month of incubation, normal human dermal fibroblasts (NHDFs) were seen growing on the scaffolds, which demonstrated that no deleterious effects were exerted by the MEW scaffolds constructed using FDM cPLA molds. The significant potential of our hybrid additive manufacturing approach to fabricate complex MEW scaffolds could be applied to a variety of tissue engineering applications, particularly in the field of vascularization. Full article

The expected outcome after a scaffold augmented hernia repair is the regeneration of a tissue composition strong enough to sustain biomechanical function over long periods. It is hypothesised that melt electrowriting (MEW) medical-grade polycaprolactone (mPCL) scaffolds loaded with platelet-rich plasma (PRP) will enhance soft tissue regeneration in fascial defects in abdominal and vaginal sheep models. A pre-clinical evaluation of vaginal and abdominal hernia reconstruction using mPCL mesh scaffolds and polypropylene (PP) meshes was undertaken using an ovine model. Each sheep was implanted with both a PP mesh (control group), and a mPCL mesh loaded with PRP (experimental group) in both abdominal and vaginal sites. Mechanical properties of the tissue-mesh complexes were assessed with plunger tests. Tissue responses to the implanted meshes were evaluated via histology, immunohistochemistry and histomorphometry. At 6 months post-surgery, the mPCL mesh was less stiff than the PP mesh, but stiffer than the native tissue, while showing equitable collagen and vascular ingrowth when compared to PP mesh. The results of this pilot study were supportive of mPCL as a safe and effective biodegradable scaffold for hernia and vaginal prolapse repair, hence a full-scale long-term study (over 24–36 months) with an adequate sample size is recommended. Full article

New additive manufacturing techniques, such as melting electro-writing (MEW) or near-field electrospinning (NFES), are now used to include microfibers inside 3D printed scaffolds as FDM printers present a limited resolution in the XY axis, not making it easy to go under 100 µm without dealing with nozzle troubles. This work studies the possibility of creating reproducible microscopic internal fibers inside scaffolds printed by standard 3D printing. For this purpose, novel algorithms generating deposition routines (G-code) based on primitive geometrical figures were created by python scripts, modifying basic deposition conditions such as temperature, speed, or material flow. To evaluate the influence of these printing conditions on the creation of internal patterns at the microscopic level, an optical analysis of the printed scaffolds was carried out using a digital microscope and subsequent image analysis with ImageJ software. To conclude, the formation of heterogeneously shaped microfilaments (48 ± 12 µm, mean ± S.D.) was achieved in a standard FDM 3D Printer with the strategies developed in this work, and it was found that the optimum conditions for obtaining such microfibers were high speeds and a reduced extrusion multiplier. Full article

The fabrication of patient-specific scaffolds for bone substitutes is possible through extrusion-based 3D printing of calcium phosphate cements (CPC) which allows the generation of structures with a high degree of customization and interconnected porosity. Given the brittleness of this clinically approved material, the stability of open-porous scaffolds cannot always be secured. Herein, a multi-technological approach allowed the simultaneous combination of CPC printing with melt electrowriting (MEW) of polycaprolactone (PCL) microfibers in an alternating, tunable design in one automated fabrication process. The hybrid CPC+PCL scaffolds with varying CPC strand distance (800–2000 µm) and integrated PCL fibers featured a strong CPC to PCL interface. While no adverse effect on mechanical stiffness was detected by the PCL-supported scaffold design; the microfiber integration led to an improved integrity. The pore distance between CPC strands was gradually increased to identify at which critical CPC porosity the microfibers would have a significant impact on pore bridging behavior and growth of seeded cells. At a CPC strand distance of 1600 µm, after 2 weeks of cultivation, the incorporation of PCL fibers led to pore coverage by a human mesenchymal stem cell line and an elevated proliferation level of murine pre-osteoblasts. The integrated fabrication approach allows versatile design adjustments on different levels. Full article

To address the increasing demand for safe and effective treatment options for pelvic organ prolapse (POP) due to the worldwide ban of the traditional polypropylene meshes, this study introduced degradable polycaprolactone (PCL)/polyethylene glycol (PEG) composite meshes fabricated with melt-electrowriting (MEW). Two PCL/PEG mesh groups: 90:10 and 75:25 (PCL:PEG, wt%) were fabricated and characterized for their degradation rate and mechanical properties, with PCL meshes used as a control. The PCL/PEG composites showed controllable degradation rates by adjusting the PEG content and produced mechanical properties, such as maximal forces, that were higher than PCL alone. The antibacterial properties of the meshes were elicited by coating them with a commonly used antibiotic: azithromycin. Two dosage levels were used for the coating: 0.5 mg and 1 mg per mesh, and both dosage levels were found to be effective in suppressing the growth of S. aureus bacteria. The biocompatibility of the meshes was assessed using human immortalized adipose derived mesenchymal stem cells (hMSC). In vitro assays were used to assess the cell viability (LIVE/DEAD assay), cell metabolic activity (alamarBlue assay) and cell morphology on the meshes (fluorescent and electron microscopy). The cell attachment was found to decrease with increased PEG content. The freshly drug-coated meshes showed signs of cytotoxicity during the cell study process. However, when pre-released for 14 days in phosphate buffered saline, the initial delay in cell attachment on the drug-coated mesh groups showed full recovery at the 14-day cell culture time point. These results indicated that the PCL/PEG meshes with antibiotics coating will be an effective anti-infectious device when first implanted into the patients, and, after about 2 weeks of drug release, the mesh will be supporting cell attachment and proliferation. These meshes demonstrated a potential effective treatment option for POP that may circumvent the issues related to the traditional polypropylene meshes. Full article

Melt-electrowriting (MEW) is an emerging method that combines electrospinning and extrusion printing, allowing the fabrication of micron-scale structures suitable for tissue engineering. Compared to other additive fabrication methods, melt-electro written structures can offer more appropriate substrates for cell culture due to filament size and mechanical characteristics of the fabricated scaffolds. In this study, polycaprolactone (PCL)/graphene composites were investigated for fabrication of micron-size scaffolds through MEW. It was demonstrated that the addition of graphene can considerably improve the processability of PCL to fabricate micron-scale scaffolds with enhanced resolution. The tensile strength of the scaffold prepared from PCL/graphene composite (with only 0.5 wt.% graphene) was proved significantly (by more than 270%), better than that of the pristine PCL scaffold. Furthermore, graphene was demonstrated to be a suitable material for tailoring the degradation process to avoid undesirable bulk degradation, rapid mass loss and damage to the internal matrix of the polymer. The findings of this study offer a promising route for the fabrication of high-resolution scaffolds with micron-scale resolution for tissue engineering. Full article

Cells interact with 3D fibrous platform topography via a nano-scaled focal adhesion complex, and more research is required on how osteoblasts sense and respond to random and aligned fibers through nano-sized focal adhesions and their downstream events. The present study assessed human primary osteoblast cells’ sensing and response to random and aligned medical-grade polycaprolactone (PCL) fibrous 3D scaffolds fabricated via the melt electrowriting (MEW) technique. Cells cultured on a tissue culture plate (TCP) were used as 2D controls. Compared to 2D TCP, 3D MEW fibrous substrates led to immature vinculin focal adhesion formation and significantly reduced nuclear localization of the mechanosensor-yes-associated protein (YAP). Notably, aligned MEW fibers induced elongated cell and nucleus shape and highly activated global DNA methylation of 5-methylcytosine, 5-hydroxymethylcytosine, and N-6 methylated deoxyadenosine compared to the random fibers. Furthermore, although osteogenic markers (osterix-OSX and bone sialoprotein-BSP) were significantly enhanced in PCL-R and PCL-A groups at seven days post-osteogenic differentiation, calcium deposits on all seeded samples did not show a difference after normalizing for DNA content after three weeks of osteogenic induction. Overall, our study linked 3D extracellular fiber alignment to nano-focal adhesion complex, nuclear mechanosensing, DNA epigenetics at an early point (24 h), and longer-term changes in osteoblast osteogenic differentiation. Full article

Meniscus injury and meniscectomy are strongly related to osteoarthritis, thus there is a clinical need for meniscus replacement. The purpose of this study is to create a meniscus scaffold with micro-scale circumferential and radial fibres suitable for a one-stage cell-based treatment. Poly-caprolactone-based scaffolds with three different architectures were made using melt electrowriting (MEW) technology and their in vitro performance was compared with scaffolds made using fused-deposition modelling (FDM) and with the clinically used Collagen Meniscus Implants^® (CMI^®). The scaffolds were seeded with meniscus and mesenchymal stromal cells (MSCs) in fibrin gel and cultured for 28 d. A basal level of proteoglycan production was demonstrated in MEW scaffolds, the CMI^®, and fibrin gel control, yet within the FDM scaffolds less proteoglycan production was observed. Compressive properties were assessed under uniaxial confined compression after 1 and 28 d of culture. The MEW scaffolds showed a higher Young’s modulus when compared to the CMI^® scaffolds and a higher yield point compared to FDM scaffolds. This study demonstrates the feasibility of creating a wedge-shaped meniscus scaffold with MEW using medical-grade materials and seeding the scaffold with a clinically-feasible cell number and -type for potential translation as a one-stage treatment. Full article

Near-field electrospinning (NFES) and melt electrowriting (MEW) are the process of extruding a fiber due to the force exerted by an electric field and collecting the fiber before bending instabilities occur. When paired with precise relative motion between the polymer source and the collector, a fiber can be directly written as dictated by preprogrammed geometry. As a result, this precise fiber control results in another dimension of scaffold tailorability for biomedical applications. In this review, biomedically relevant polymers that to date have manufactured fibers by NFES/MEW are explored and the present limitations in direct fiber writing of standardization in published setup details, fiber write throughput, and increased ease in the creation of complex scaffold geometries are discussed. Full article

Scaffolds made of biodegradable biomaterials are widely used to guide bone regeneration. Commonly, in vitro assessment of scaffolds’ osteogenesis potential has been performed predominantly in monoculture settings. Hence, this study evaluated the potential of an unstimulated, growth factor-free co-culture system comprised of osteoblasts (OB) and peripheral blood mononuclear cells (PBMC) over monoculture of OB as an in vitro platform for screening of bone regeneration potential of scaffolds. Particularly, this study focuses on the osteogenic differentiation and mineralized matrix formation aspects of cells. The study was performed using scaffolds fabricated by means of a melt electrowriting (MEW) technique made of medical-grade polycaprolactone (PCL), with or without a surface coating of calcium phosphate (CaP). Qualitative results, i.e., cell morphology by fluorescence imaging and matrix mineralization by von Kossa staining, indicated the differences in cell behaviours in response to scaffolds’ biomaterial. However, no obvious differences were noted between OB and OB+PBMC groups. Hence, quantitative investigation, i.e., alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP) activities, and gene expression were quantitatively evaluated by reverse transcription-polymerase chain reaction (RT-qPCR), were evaluated only of PCL/CaP scaffolds cultured with OB+PBMC, while PCL/CaP scaffolds cultured with OB or PBMC acted as a control. Although this study showed no differences in terms of osteogenic differentiation and ECM mineralization, preliminary qualitative results indicate an obvious difference in the cell/non-mineralized ECM density between scaffolds cultured with OB or OB+PBMC that could be worth further investigation. Collectively, the unstimulated, growth factor-free co-culture (OB+PBMC) system presented in this study could be beneficial for the pre-screening of scaffolds’ in vitro bone regeneration potential prior to validation in vivo. Full article