13:00   Tissue Engineering
Printability of double network alginate-based hydrogel for 3D bio-printed vascularization techniques
Immacolata Greco
Abstract: Aims: Over the past years, 3D bioprinting has emerged as a key technology in tissue engineering. Although many studies have demonstrated the possibility of creating self-sustained hollow structures in specific experimental conditions, many limitations remain in printing complex vascular networks. Vascularization is the process of creating tubular structures suitable for the transport of nutrients and oxygen to the cells. In 3D bioprinting, this natural occurring process is difficult to reproduce if one considers the number of phenomena – diffusion, gas exchange, and cells adhesion – involved. The quest for a functioning vascular structure is still open and depends on the choice of a suitable biocompatible material. Since their discovery, hydrogels are recognised as a material having great potential in different biomedical fields such as tissue engineering, drug delivery and wound healing, mainly due to their unique properties such as high-water content, biocompatibility, the possibility of functionalization and possibility to be used as scaffolds and as bio-inks. This work aims to develop a strategy to 3D bio-print self-standing vascular structures, suitable for space experiments. Methods: The printing process was carried out using an extrusion-based bio printer (Regemat 3D BIO V1). In this study, UV light was added to achieve polymerization. This combination (i.e., Regemant + UV) allows the subsequent printing of multiple materials. The hydrogel solution was exposed to UV light during the printing process to crosslinking the Poly(ethylene glycol)diacrylate (PEGDA) and thus fix the desired structure (formability). After printing, the structures were immersed in calcium chloride solution (CaCl_2) for crosslinking of the Sodium Alginate (SA). Four different hydrogel solutions were tested, and the settings of the printer were optimized. The shape fidelity of the structures was then assessed via a microscope. Results: The best glycerol concentration to achieve an appropriate viscosity was 25 % of the solution. In addition, this concentration resulted in the best line width. Conclusion: With this work, the appropriate printer configuration and hydrogel solution were identified. These results are a step forward towards the bioprinting of tubular structures that enhance cell viability. With this research, insights into the in-space manufacturing of functional tissues can be unveiled.
Nanoimprinting for high-throughput replication of geometrically precise pillars to regulate cell response
Mahya Ganjian, Khashayar Modaresifar, Dionysios Rompolas, Lidy E. Fratila-Apachitei, Amir A. Zadpoor
Abstract: Keywords: cell-pattern interactions, soft lithography, nanoimprint lithography Developing high-throughput submicron/nanopatterning fabrication techniques that also allow for a precise control over the dimensions of fabricated features is essential to enhance the efficacy of studies on cell-pattern interactions. Here, we developed a process to fulfill these two criteria. Firstly, we used electron-beam lithography (EBL) to fabricate precisely controlled arrays of submicron pillars with varying interspacing (700 nm and 1000 nm) on fused silica substrate. The interactions of MC3T3-E1 cells with these pillars were investigated. Varying interspacing was observed to significantly affect the settling state of the cells as well as their morphological characteristics (cell area and aspect ratio), actin fibers organization, and formation of focal adhesions. Interestingly, the differences were not limited to the early response of the cells to the patterned surface: while the cells possessed a smaller area on the patterns compared to the flat surface, the expression of osteopontin (OPN) significantly increased on the patterns, indicating the potential of the pillars for inducing osteogenic differentiation. Such EBL patterns were thereafter used as mother molds in the two subsequent processing steps namely soft lithography and thermal nanoimprint lithography. Following the optimization of the molding parameters of the two different steps (e.g., process time and temperature for soft lithography method and applied force, molding and de-molding temperature, time, resist thickness, etc. for nanoimprint process) the sizes of the submicron pillars as well as their interspacing were reproduced with high fidelity without damaging the mother mold (error < 20 % for the diameter all over the patterned area and only 1% error for interspacing). The method proposed in this study enables precise fabrication of submicron patterns on a wide variety of substrate materials relevant for systematic cell studies.
Alignment of keratocytes and deposited matrices in concave, micropatterned environments
Cas van der Putten, Thomas Woud, Gitta Buskermolen, Maike Werner, Carlijn Bouten, Nicholas Kurniawan
Abstract: All tissues inside the human body are complex assemblies of cells and extracellular matrix (ECM). In the aim to engineer tissues with native structure and functionality, it is important to control the behavior of cells and deposited ECM. To do so, cells are often subjected to individual in vitro environmental cues mimicked from the in vivo ECM, for example contact- or curvature guidance cues. Although this approach gives useful insights in cell behavior, the in vivo environment presents a plethora of cues at the same time. Here we present a novel experimental platform to explore cell behavior to the combination of contact- and curvature guidance cues. Contact guidance cues are applied using UV-photopatterning of ECM proteins to a culture chip containing a library of convex and concave curvatures mimicking tissue geometry [1]. When myofibroblasts and endothelial cells are subjected to opposing ECM patterns and concave, cylindrical curvatures, the cells show an alignment response in the direction of the protein pattern. On convex curvatures, however, myofibroblasts reorient to avoid a bent morphology, consistent with the results using curvature guidance cues only. Using this approach, we now can not only investigate cell behavior in response to more representative environments, but also use this knowledge with the aim to steer cell alignment and eventually the orientation of newly produced matrix. Using corneal fibroblasts on patterned spherical pits of varying curvature (1/8000 µm-1 ≤ ĸ ≤ 1/250 µm-1), we quantified the orientation of cells and newly produced collagen in these complex environments. After 1 day, cell orientation is predominantly influenced by the protein pattern, completely in line with the results of the previously tested cells. After 7 days, the orientation of both cells and collagens shifted away from the direction of the protein patterns. In pits with ĸ < 1/1000 µm-1, aligned sheets of cells and collagen were formed, whereas curvature avoidance behavior is dominant in pits with ĸ ≥ 1/1000 µm-1. With these results we not only get a better understanding of cell behavior in complex environments, but also gain knowhow on steering cell and collagen orientation in the context of tissue engineering. [1] van der Putten et al., ACS Appl. Mater. Interfaces, 2021
Antibacterial and osteoinductive resorbable MgONPs/PTH coaxial electrospinning barrier membrane for guided bone regeneration
yiwen dong, jinsong liu
Abstract: The key target for this study is to manufacture a novel barrier membrane can fit the bacteriostatic, osteoinductive and resorbable propertites for clinical needs. Hence, magnesium oxide nanoparticles (MgONPs) and parathyroid hormone (PTH) were combined individually with polycaprolactone (PCL) to manufacture a shell/core nanofiber membrane using coaxial electrospinning to fabricate a dual-drug-release membrane for the first attempt. The multifunctional MgONPs/PTH barrier membrane has outstanding antibacterial potential against Escherichia coli and Staphylococcus aureus by the long-term release of MgONPs, which may contribute to bone regeneration for the decreasing incidence of postoperative infection. Furthermore, exceptional osteoinductive propertivies of the membrane with the assist of high dosage PTH both in vivo and in vitro indicated the possibility for higher quality and more quantity of new bone. Furthermore, we discovered that the incorporation of MgONPs significantly extand PTH’s release which is a good signal for bone regeneration as well. Therefore, we believe that the unique barrier membrane can provide a beneficial opportunity for bone regeneration in the defect area.
Optimization of chondrocyte-derived ECM mechanical properties for cartilage regeneration
Alejandro Reina Mahecha, Theo G. van Kooten, Inge S. Zuhorn, Prashant K. Sharma
Abstract: Osteoarthritis (OA) is a multifactorial degenerative joint disease. It is a pathology that promotes cartilage degeneration due to pro-inflammatory cytokines and proteases in the synovial fluid, secreted by the cartilage's resident cells, i.e., chondrocytes [1, 2]. OA creates a homeostatic imbalance that, combined with the high mechanical demand, makes tissue regeneration a remaining challenge, especially in lower limb joints. While the current treatments are highly invasive and not always beneficial for the patient, a solution to stop disease progression and trigger articular cartilage regeneration is needed. Current tissue engineering approaches fail to mimic the tissue's mechanical properties resulting in fibrocartilage formation [3]. Here we explored the potential of embryoid bodies (EBs) generated from pluripotent stem cells to induce cartilage's extracellular matrix (ECM) with similar mechanical properties as native articular cartilage. In order to enhance reproducibility, the size homogeneity of EBs is important and was accomplished by using a custom-made microwell system. The EB size uniformity is expected to diminish variation in cell mechanics as well as maximize nutrient availability throughout the EBs, making them an ideal starting point for cell differentiation [3]. Chondrocyte differentiation was induced in EBs using a chondrogenic defined medium (CDM) containing essential chondrogenic growth factors (TGFß3, BM2, and GDF5) [4, 5]. After differentiation of the cell aggregates for 21 days, the produced/deposited ECM contained collagen II and glycosaminoglycans, indicators of articular cartilage formation. The tissue stiffness of differentiated chondrocytes, measured using a Low Load Compression Testor (LLCT), after 21 days in CDM was similar to native articular cartilage, making this approach suitable for studying cartilage regeneration for OA treatment by, e.g., activation of regenerative pathways.
Thin, flexible and porous polydimethylsiloxane scaffold for barrier tissue culture models
Mariia Zakharova, Elsbeth Bossink, Kerensa Broersen, Andries van der Meer, Loes Segerink
Abstract: Commercially available track-etched 10 μm thick polymer membranes have been intensively used for studying cellular barriers in cell culture models. However, their thickness and rigidity hampers direct cell-cell interaction between the cells cultured on both sides of the mebrane and offer limited functionality. Previously reported polydimethylsiloxane (PDMS) membranes are 4 µm thick, are transparent allowing for optical microscopic visualization. Moreover, the flexibility of such membranes allows the application of physiologically relevant cyclic strain on the cell layers. To further facilitate interaction between cell layers positioned at opposite sides of the membrane, we have developed a PDMS membrane that is 2 μm thick with defined pore size and distribution and studied its applicability as a cell-culture substrate. While the detailed fabrication protocol of these membranes has been reported by us [1], here we will present the possible applications of these membranes. We found that, after fabrication, the membranes can be either transferred to a PDMS-based chip or can be glued to Transwell inserts [1]. For transfer to a PDMS-based chip, an oxygen plasma treatment can be applied to bond the chip to the PDMS membrane. In this way, we successfully transferred the membranes in multiplexed cell culture chips and mimicked the Blood-Brain barrier (BBB) by culturing hCMEC/D3 endothelial cells and astrocytes on the 5 μm pore-sized membranes [2]. We also showed the integration of the membranes inside the Transwell inserts [1], by applying PDMS glue to bond the membrane to the rim of the insert. We showed that the reduced thickness of the membrane improved the cell-cell interactions in the BBB model [1]. Additionally, we were able to fabricate membranes with a larger pore diameter of 10 μm that can be used for studying cell extravasation. Finally, tissue culture models of organs such as the lung and gastrointestinal tract require the possibility to stretch the cells. Therefore, by applying the maximum reachable negative pressure of -100 kPa to both sides of the membrane in a microfluidic cell culture chip, we showed that it can be deformed up to, but not limited to, 85% and remain intact. The strain correlates linearly with the pressure, leading to 10% deformation for every -10kPa applied following this trend up to -70 kPa. Moreover, due to the low thickness of the membrane, less pressure is needed to deform the membrane compared to previous reports [3]. In conclusion, we explored the applications of 2 μm thick PDMS membranes and demonstrate it can be used as an alternative scaffold to commercially available track-etched membranes.

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