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Employment of Melt Electrowriting for the design of regenerative graftsvon Witzleben, Max 01 July 2024 (has links)
BACKGROUND Cell-sized structures such as electrospun mats have been shown to tailor cell growth in a variety of ways and thus have great potential in the development of regenerative implants. Due to their thinness of several hundreds of micrometers, these mats mostly act as coatings on larger matrices, but the use of cytotoxic solvents complicates the translational process. A relatively new technique, melt electrowriting (MEW), offers similar properties but relinquishes cytotoxic solvents. Instead, thermoplastics such as polycaprolactone (PCL) are melted and processed under high voltage to form fibers with a precise fiber diameter that can be deposited in highly ordered meshes using a three-axis system. Outcome of the MEW processes are fiber architectures with defined fiber diameter, fiber spacing and tailored porosity within the cellular dimensions. This contrasts with previous electrospun mats, which mostly exhibit chaotic fiber architectures. RESEARCH QUESTIONS Due to the novelty of MEW, all studies so far used highly customized laboratory printers to produce MEW membranes, complicating translation to the clinic. Therefore, one of the first commercial MEW printer had to be established and the printing characteristics needed to be found to maintain homogeneous fiber diameters throughout the printing process and to investigate the compatibility with other printing techniques. Large tympanic membrane defects, such as those caused by chronic otitis media and other conditions, are currently closed with autologous materials in an elaborate procedure that may result in side effects such as hearing loss. Customized MEW meshes could improve this situation if they demonstrate similar mechanical and vibrational properties as the TM or the respective autologous materials. To this end, the variety of different design parameters such as fiber diameter, fiber spacing, layer-to-layer orientation, and number of layers should be investigated. Ideally, the collagen fiber structure and the curvature of the TM could be mimicked, too. Furthermore, the behavior of TM-typical cells such as keratinocytes and fibroblasts on the artificial scaffolds should be analyzed. This should simulate a potential regrowth of the TM collagen structure in vivo by migrating cells from the surrounding tissue onto the artificial membranes. Larger regenerative implant materials produced by other (additive) manufacturing processes do not offer geometries within cell dimension, therefore a combination with MEW could facilitate the scaffold-cell interaction. Hence, the objective of the present work was to investigate to what extent MEW can be combined with other additive manufacturing processes and what effects combined implant materials have on cell adhesion, alignment, and migration. MATERIALS & METHODS A GeSiM Printer 3.1. equipped with a MEW module was used for printing. Medical grade PCL was utilized to produce artificial membranes. Medical grade collagen type I was applied to create airtight membranes and improve cell compatibility. All scaffolds were mechanically tested using a uniaxial testing machine to characterize either the bending stiffness of the artificial TM grafts or the Young's modulus of the bone and tendon grafts. Compression tests were performed on the bone grafts, whereas tensile tests were used to evaluate the tendon grafts. Vibration analysis of the TM grafts was performed in collaboration at the Ear Clinic of Dresden University Hospital. For in vitro analyses of the TM grafts, immortalized and primary keratinocytes (HaCaT, HEKn) and primary fibroblasts (NHDF) were seeded on the grafts and analyzed. Murine calvarial osteoblast progenitor cells (MC3T3-E1) and immortalized human mesenchymal stem cells (hTERT-MSC) were seeded on the calcium phosphate cement and PCL composites. Adipose-derived mesenchymal stem cells (AT-MSC) were seeded onto the PLA-PCL tendon grafts. Standardized biochemical assays and fluorescence microscopy were used to analyze cell behavior.
RESULTS Specific scaffold designs were developed to create a TM graft with comparable mechanical and similar vibrational properties to the native TM. In addition, the constructs showed high cell compatibility. Circular and radial fibers were integrated to mimic the native collagen structure closer. The combination of MEW with extrusion printing of sacrificial pyramids allowed for resembling the curvature of the TM. Overall, these adjustments minimized the gap between implant and native TM. By increasing the number of layers, the yield strength of the TM could be increased, but with a (small) decrease in vibration properties. A co-culture of primary fibroblasts and keratinocytes mimicked the in vivo migration of these cell types on the scaffolds so that the native collagen architecture could be restored after implantation in vivo (Publication I + II). For the first time, the bone graft material calcium phosphate cement (CPC) has been combined with PCL microfibers in a single fabrication process by combining MEW and extrusion printing (Publication III). Geometries in clinically relevant defect sizes of up to 3 cm with a variety of different pore structures were realized. The microporosity thus created within the macroporosity of the CPC structures had no significant effect on the cell growth closing these pores compared to CPC scaffolds without microfibers. From a mechanical point of view, the microfibers did not affect the adhesion between the CPC layers but fixed the CPC fragments during and after mechanical loading, so that the PCL-reinforced CPC scaffolds did not splinter in contrast to the pure CPC structures. When the PCL mats were printed wider than the CPC scaffolds, the protruding mesh provided an additional fixation option for the composite scaffolds in a potential defect area during surgery.
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