3D Humanized Bioprinted Tubulointerstitium Model to Emulate Renal Fibrosis In Vitro.

Chronic kidney disease (CKD) leads to a gradual loss of kidney function, with fibrosis as pathological endpoint, which is characterized by extracellular matrix (ECM) deposition and remodeling. Traditionally, in vivo models are used to study interstitial fibrosis, through histological characterization of biopsy tissue. However, ethical considerations and the 3Rs (replacement, reduction, and refinement) regulations emphasizes the need for humanized 3D in vitro models. This study introduces a bioprinted in vitro model which combines primary human cells and decellularized and partially digested extracellular matrix (ddECM). A protocol was established to decellularize kidney pig tissue and the ddECM was used to encapsulate human renal cells. To investigate fibrosis progression, cells were treated with transforming growth factor beta 1 (TGF-ß1), and the mechanical properties of the ddECM hydrogel were modulated using vitamin B2 crosslinking. The bioprinting perfusable model replicates the renal tubulointerstitium. Results show an increased Young's modulus over time, together with the increase of ECM components and cell dedifferentiation toward myofibroblasts. Multiple fibrotic genes resulted upregulated, and the model closely resembled fibrotic human tissue in terms of collagen deposition. This 3D bioprinted model offers a more physiologically relevant platform for studying kidney fibrosis, potentially improving disease progression research and high-throughput drug screening.

Is There an Interplay between Environmental Factors, Microbiota Imbalance, and Cancer Chemotherapy-Associated Intestinal Mucositis?

Interindividual variation in drug efficacy and toxicity is a significant problem, potentially leading to adverse clinical and economic public health outcomes. While pharmacogenetics and pharmacogenomics have long been considered the primary causes of such heterogeneous responses, pharmacomicrobiomics has recently gained attention. The microbiome, a community of microorganisms living in or on the human body, is a critical determinant of drug response and toxicity. Factors such as diet, lifestyle, exposure to xenobiotics, antibiotics use, illness, and genetics can influence the composition of the microbiota. Changes in the intestinal microbiota are particularly influential in drug responsiveness, especially in cancer chemotherapy. The microbiota can modulate an individual's response to a drug, affecting its bioavailability, clinical effect, and toxicity, affecting treatment outcomes and patient quality of life. For instance, the microbiota can convert drugs into active or toxic metabolites, influencing their efficacy and side effects. Alternatively, chemotherapy can also alter the microbiota, creating a bidirectional interplay. Probiotics have shown promise in modulating the microbiome and ameliorating chemotherapy side effects, highlighting the potential for microbiota-targeted interventions in improving cancer treatment outcomes. This opinion paper addresses how environmental factors and chemotherapy-induced dysbiosis impact cancer chemotherapy gastrointestinal toxicity.

Matrix metalloproteinase degradable, in situ photocrosslinked nanocomposite bioinks for bioprinting applications.

The development of suitable bioinks with high printability, mechanical strength, biodegradability, and biocompatibility is a key challenge for the clinical translation of 3D constructs produced with bioprinting technologies. In this work, we developed a new type of nanocomposite bioinks containing thiolated mesoporous silica nanoparticles (MSN) that act as active fillers within norbornene-functionalized hydrogels. The MSNs could rapidly covalently crosslink the hydrogels upon exposure to UV light. The mechanical properties of the gels could be modulated from 9.3 to 19.7 kPa with increasing concentrations of MSN. The ability of the MSN to covalently crosslink polymeric networks was, however, significantly influenced by polymer architecture and the number of functional groups. Modification of the outer surface of MSNs with matrix metalloproteinase (MMP) sensitive peptides (MSN-MMPs) resulted in proteinase K and MMP-9 enzyme responsive biodegradable bioinks. Additional cysteine modified RGD peptide incorporation enhanced cell-matrix interactions and reduced the gelation time for bioprinting. The nanocomposite bioinks could be printed by using extrusion-based bioprinting. Our nanocomposite bioinks preserved their shape during in vitro studies and encapsulated MG63 cells preserved their viability and proliferated within the bioinks. As such, our nanocomposite bioinks are promising bioinks for creating bioprinted constructs with tunable mechanical and degradation properties.

Comparison of Zinc Oxide Nanoparticle Integration into Non-Woven Fabrics Using Different Functionalisation Methods for Prospective Application as Active Facemasks.

The development of advanced facemasks stands out as a paramount priority in enhancing healthcare preparedness. In this work, different polypropylene non-woven fabrics (NWF) were characterised regarding their structural, physicochemical and comfort-related properties. The selected NWF for the intermediate layer was functionalised with zinc oxide nanoparticles (ZnO NPs) 0.3 and 1.2wt% using three different methods: electrospinning, dip-pad-dry and exhaustion. After the confirmation of ZnO NP content and distribution within the textile fibres by morphological and chemical analysis, the samples were evaluated regarding their antimicrobial properties. The functionalised fabrics obtained via dip-pad-dry unveiled the most promising data, with 0.017 ± 0.013wt% ZnO NPs being mostly located at the fibre's surface and capable of total eradication of Staphylococcus aureus and Escherichia coli colonies within the tested 24 h (ISO 22196 standard), as well as significantly contributing (**** p < 0.0001) to the growth inhibition of the bacteriophage MS2, a surrogate of the SARS-CoV-2 virus (ISO 18184 standard). A three-layered structure was assembled and thermoformed to obtain facemasks combining the previously chosen NWF, and its resulting antimicrobial capacity, filtration efficiency and breathability (NP EN ISO 149) were assessed. The developed three-layered and multiscaled fibrous structures with antimicrobial capacities hold immense potential as active individual protection facemasks.

Flexible, Suturable, and Leak-free Scaffolds for Vascular Tissue Engineering Using Melt Spinning.

Coronary artery disease affects millions worldwide. Bypass surgery remains the gold standard; however, autologous tissue is not always available. Hence, the need for an off-the-shelf graft to treat these patients remains extremely high. Using melt spinning, we describe here the fabrication of tubular scaffolds composed of microfibers aligned in the circumferential orientation mimicking the organized extracellular matrix in the tunica media of arteries. By variation of the translational extruder speed, the angle between fibers ranged from 0 to ∼30°. Scaffolds with the highest angle showed the best performance in a three-point bending test. These constructs could be bent up to 160% strain without kinking or breakage. Furthermore, when liquid was passed through the scaffolds, no leakage was observed. Suturing of native arteries was successful. Mesenchymal stromal cells were seeded on the scaffolds and differentiated into vascular smooth muscle-like cells (vSMCs) by reduction of serum and addition of transforming growth factor beta 1 and ascorbic acid. The scaffolds with a higher angle between fibers showed increased expression of vSMC markers alpha smooth muscle actin, calponin, and smooth muscle protein 22-alpha, whereas a decrease in collagen 1 expression was observed, indicating a positive contractile phenotype. Endothelial cells were seeded on the repopulated scaffolds and formed a tightly packed monolayer on the luminal side. Our study shows a one-step fabrication for ECM-mimicking scaffolds with good handleability, leak-free property, and suturability; the excellent biocompatibility allowed the growth of a bilayered construct. Future work will explore the possibility of using these scaffolds as vascular conduits in in vivo settings.

Manufacturing of scaffolds with interconnected internal open porosity and surface roughness.

Manufacturing of three-dimensional scaffolds with multiple levels of porosity are an advantage in tissue regeneration approaches to influence cell behavior. Three-dimensional scaffolds with surface roughness and intra-filament open porosity were successfully fabricated by additive manufacturing combined with chemical foaming and porogen leaching without the need of toxic solvents. The decomposition of sodium citrate, a chemical blowing agent, generated pores within the scaffold filaments, which were interconnected and opened to the external environment by leaching of a water-soluble sacrificial phase, as confirmed by micro-CT and buoyancy measurements. The additional porosity did not result in lower elastic modulus, but in higher strain at maximum load, i.e. scaffold ductility. Human mesenchymal stromal cells cultured for 24 h adhered in greater numbers on these scaffolds when compared to plain additive-manufactured ones, irrespectively of the scaffold pre-treatment method. Additionally, they showed a more spread and random morphology, which is known to influence cell fate. Cells cultured for a longer period exhibited enhanced metabolic activity while secreting higher osteogenic markers after 7 days in culture. STATEMENT OF SIGNIFICANCE: Inspired by the function of hierarchical cellular structures in natural materials, this work elucidates the development of scaffolds with multiscale porosity by combining in-situ foaming and additive manufacturing, and successive porogen leaching. The resulting scaffolds displayed enhanced mechanical toughness and multiscale pore network interconnectivity, combined with early differentiation of adult mesenchymal stromal cells into the osteogenic lineage.

Assessment of motor function in individuals with hemiplegia post-stroke / Evaluación de la función motriz en hemipléjicos posaccidente cerebrovascular / Avaliação da função motora em hemiplégicos pós-acidente vascular encefálico

ABSTRACT This study aims to compare the performance of the sit-to-stand test and walking speed in individuals with chronic hemiplegia post-stroke and a control group (CG). Moreover, we will investigate whether lower limb resistance, measured based on the sit-to-stand test, is related to walking speed in individuals with chronic hemiplegia and a CG. Finally, we will verify if there are intra-group differences for the tests by dividing the hemiplegia group (HG) according to motor and sensorimotor function assessment classification. A cross-sectional design was used among a group with chronic hemiplegia (n=28) and a healthy CG (n=22). The HG was classified by the Fugl-Meyer scale, and both groups were evaluated using the 1-minute sit-to-stand test. The walking speed was calculated using a 3D kinematics system. Lower limb resistance among HG differed significantly from the CG, as well as walking speed. We found a strong correlation between the tests (ρ=0.773; p<0.001). No differences were found for the sit-to-stand tests and walking speed when dividing the HG into individuals with greater or lesser motor and sensory impairment, using the Fugl-Meyer scale. Therefore, individuals with hemiplegia, regardless of having a more pronounced classification of motor and sensory impairment on the Fugl-Meyer scale, showed lower limb resistance and lower walking speed compared with individuals without hemiplegia post-stroke.

RESUMEN El objetivo de este estudio es comparar el desempeño del test de levantarse y sentarse y la velocidad de marcha en individuos con hemiplejía crónica debido a accidente cerebrovascular (ACV) y un grupo control (GC). Además, se investigará si existe asociación entre la resistencia de los miembros inferiores, medida desde el test de levantarse y sentarse, y la velocidad de marcha en individuos con hemiplejía crónica y un GC. Por último, se verificará si existen diferencias intragrupales en las pruebas al dividir el grupo hemiplejía (GH) según la clasificación de evaluación de deterioro motor y sensorial. El método utilizado fue el transversal en un grupo con hemiplejía crónica (n=28) y un GC sin ninguna patología (n=22). El GH se clasificó mediante la escala de Fugl-Meyer, y ambos grupos se evaluaron mediante el test de levantarse y sentarse de un minuto. La velocidad de marcha se calculó mediante el sistema cinemático tridimensional. Entre los resultados obtenidos, se observó que la resistencia de los miembros inferiores entre GH difería significativamente del GC, así como la velocidad de marcha. Se demostró una fuerte correlación entre las pruebas (ρ=0,773; p<0,001). No se encontraron diferencias en las pruebas de levantarse y sentarse y la velocidad de la marcha al dividir el GH en individuos con mayor o menor deterioro motor y sensorial, utilizando la escala de Fugl-Meyer. Por lo tanto, las personas con hemiplejía, independientemente de tener un mayor deterioro motor y sensorial según la escala de Fugl-Meyer, tuvieron una menor resistencia de las extremidades inferiores y una menor velocidad de marcha en comparación con las personas sin hemiplejía pos-ACV.

RESUMO O objetivo deste estudo é comparar os desempenhos no teste de sentar e levantar e a velocidade de caminhada de indivíduos com hemiplegia crônica decorrente de acidente vascular encefálico (AVE) e um grupo-controle (GC). Além disso, será investigado se existe associação entre a resistência de membros inferiores, mensurada a partir do teste de sentar e levantar, e a velocidade de caminhada em indivíduos com hemiplegia crônica e um GC. Por fim, será verificado se existem diferenças intragrupo para os testes ao dividir o grupo hemiplegia (GH) de acordo com a classificação de avaliação do comprometimento motor e sensorial. O método utilizado foi o delineamento transversal entre um grupo com hemiplegia crônica (n=28) e um GC sem nenhuma patologia (n=22). O GH foi classificado a partir da escala de Fugl-Meyer, e ambos os grupos foram avaliados por meio do teste de sentar e levantar de um minuto. A velocidade de caminhada foi calculada a partir de um sistema de cinemetria tridimensional. Entre os resultados obtidos, foi percebido que a resistência de membros inferiores do GH diferiu significativamente do GC, assim como a velocidade de caminhada. Foi demonstrada uma correlação forte entre os testes (ρ=0,773; p<0,001). Não foram encontradas diferenças nos testes de sentar e levantar e velocidade de caminhada ao dividir o GH em indivíduos com maior ou menor comprometimento motor e sensorial, com a escala de Fugl-Meyer. Portanto, indivíduos com hemiplegia, independentemente de ter uma classificação de comprometimento motor e sensorial mais acentuada na escala de Fugl-Meyer, apresentaram menor resistência de membros inferiores e menor velocidade de caminhada comparados com indivíduos sem hemiplegia pós-AVE.

Effect of high content nanohydroxyapatite composite scaffolds prepared via melt extrusion additive manufacturing on the osteogenic differentiation of human mesenchymal stromal cells.

The field of bone tissue engineering seeks to mimic the bone extracellular matrix composition, balancing the organic and inorganic components. In this regard, additive manufacturing (AM) of high content calcium phosphate (CaP)-polymer composites holds great promise towards the design of bioactive scaffolds. Yet, the biological performance of such scaffolds is still poorly characterized. In this study, melt extrusion AM (ME-AM) was used to fabricate poly(ethylene oxide terephthalate)/poly(butylene terephthalate) (PEOT/PBT)-nanohydroxyapatite (nHA) scaffolds with up to 45 wt% nHA, which presented significantly enhanced compressive mechanical properties, to evaluate their in vitro osteogenic potential as a function of nHA content. While osteogenic gene upregulation and matrix mineralization were observed on all scaffold types when cultured in osteogenic media, human mesenchymal stromal cells did not present an explicitly clear osteogenic phenotype, within the evaluated timeframe, in basic media cultures (i.e. without osteogenic factors). Yet, due to the adsorption of calcium and inorganic phosphate ions from cell culture media and simulated body fluid, the formation of a CaP layer was observed on PEOT/PBT-nHA 45 wt% scaffolds, which is hypothesized to account for their bone forming ability in the long term in vitro, and osteoconductivity in vivo.

Development of a device useful to reproducibly produce large quantities of viable and uniform stem cell spheroids with controlled diameters.

Three-dimensional cellular aggregates can mimic the natural microenvironment of tissues and organs and obtaining them through controlled and reproducible processes is mandatory for scaling up and implementing drug cytotoxicity and efficacy tests, as well as tissue engineering protocols. The purpose of this work was to develop and evaluate the performance of a device with two different geometries fabricated by additive manufacturing. The methodology was based on casting a microwell array insert using a non-adhesive hydrogel to obtain highly regular microcavities to standardize spheroid formation and morphology. Spheroids of dental pulp stem cells, bone marrow stromal cells and embryonic stem cells showing high cell viability and average diameters of around 253, 220, and 500 µm, respectively, were produced using the device with the geometry considered most adequate. The cell aggregates showed sphericity indexes above 0.9 and regular surfaces (solidity index higher than 0.96). Around 1000 spheroids could be produced in a standard six-well plate. Overall, these results show that this method facilitates obtaining a large number of uniform, viable spheroids with pre-specified average diameters and through a low-cost and reproducible process for a myriad of applications.

Avaliação do controle postural em praticantes de dança de salão / Evaluation of postural control in practitioners of ballroom dancing

Este estudo teve como objetivo analisar o efeito imediato da prática de dança de salão sobre o controle postural estático de seus praticantes, juntamente aos fatores que possam influenciá-lo. Optou-se por um estudo transversal de caráter observacional quantitativo em uma amostra por conveniência, constituída por 19 homens e 19 mulheres praticantes de dança de salão, que participaram de uma prática de dança de 120 minutos. Foram coletados os dados sociodemográficos, antropométricos e sintomas osteomusculares. Para a avaliação do centro de pressão (COP), foi utilizado uma plataforma de força antes e depois da prática. O único fator que demonstrou diferença significativa no controle postural antes da prática de dança foi o sexo, sendo os homens que apresentam maiores deslocamentos no COP. Comparando o efeito da prática de 120 minutos de dança, em todos os participantes, houve uma diminuição do deslocamento do COP na posição bipodal no COPap (p= 0.028) e COPvel (p= 0.003), na posição unipodal COPvel (p= 0.006) e na posição semi-tandem COPml (p= 0.026). O efeito imediato de uma prática de dança de salão contribui para o controle postural em ambos os sexos. (AU)

This study aimed to analyze the immediate effect of the practice ballroom dancing on the static postural control of its practitioners, along with factors that can influence it. We opted for a cross-sectional study of quantitative observational in a sample of convenience of 19 men and 19 women practitioner's ballroom dancing, who participated in a dance practice about 120 minutes. We collected sociodemographic data, anthropometric and musculoskeletal symptoms. For the evaluation of center of pressure (COP), was used a force platform before and after practice. The only factor that demonstrated a significant association with postural control prior to dance practice was the genre, men show greater displacements at the COP. Comparing the effect of the practice of 120 minutes of dance, in all participants, a decrease in COP displacement in the bipedal position in COPap (p = 0.028) and COPvel (p = 0.003), in the single-leg position COPvel (p = 0.006) and in the semi-tandem position COPml (p = 0.026). The immediate effect of a ballroom practice contributes to postural control in both sexes. (AU)

Study of the Filtration Performance of Multilayer and Multiscale Fibrous Structures.

As the incidence of small-diameter particles in the air has increased in recent decades, the development of efficient filtration systems is both urgent and necessary. Nanotechnology, more precisely, electrospun nanofibres, has been identified as a potential solution for this issue, since it allows for the production of membranes with high rates of fibres per unit area, increasing the probability of nanoparticle collision and consequent retention. In the present study, the electrospinning technique of polyamide nanofibre production was optimized with the variation of parameters such as polymer concentration, flow rate and needle diameter. The optimized polyamide nanofibres were combined with polypropylene and polyester microfibres to construct a multilayer and multiscale system with an increased filtration efficiency. We observed that the penetration value of the multilayer system with a PA membrane in the composition, produced for 20 min in the electrospinning, is 2.7 times smaller than the penetration value of the system with the absence of micro and nano fibers.

Bioprinting of kidney in vitro models: cells, biomaterials, and manufacturing techniques.

The number of patients with end-stage renal disease is continuously increasing worldwide. The only therapies for these patients are dialysis and organ transplantation, but the latter is limited due to the insufficient number of donor kidneys available. Research in kidney disease and alternative therapies are therefore of outmost importance. In vitro models that mimic human kidney functions are essential to provide better insights in disease and ultimately novel therapies. Bioprinting techniques have been increasingly used to create models with some degree of function, but their true potential is yet to be achieved. Bioprinted renal tissues and kidney-like constructs presents challenges, for example, choosing suitable renal cells and biomaterials for the formulation of bioinks. In addition, the fabrication of complex renal biological structures is still a major bottleneck. Advances in pluripotent stem cell-derived renal progenitors has contributed to in vivo-like rudiment structures with multiple renal cells, and these started to make a great impact on the achieved models. Natural- or synthetic-based biomaterial inks, such as kidney-derived extracellular matrix and gelatin-fibrin hydrogels, which show the potential to partially replicate in vivo-like microenvironments, have been largely investigated for bioprinting. As the field progresses, technological, biological and biomaterial developments will be required to yield fully functional in vitro tissues that can contribute to a better understanding of renal disease, to improve predictability in vitro of novel therapeutics, and to facilitate the development of alternative regenerative or replacement treatments. In this review, we resume the main advances on kidney in vitro models reported so far.

Cell spheroids as a versatile research platform: formation mechanisms, high throughput production, characterization and applications.

Three-dimensional (3D) cell culture has tremendous advantages to closely mimic thein vivoarchitecture and microenvironment of healthy tissue and organs, as well as of solid tumors. Spheroids are currently the most attractive 3D model to produce uniform reproducible cell structures as well as a potential basis for engineering large tissues and complex organs. In this review we discuss, from an engineering perspective, processes to obtain uniform 3D cell spheroids, comparing dynamic and static cultures and considering aspects such as mass transfer and shear stress. In addition, computational and mathematical modeling of complex cell spheroid systems are discussed. The non-cell-adhesive hydrogel-based method and dynamic cell culture in bioreactors are focused in detail and the myriad of developed spheroid characterization techniques is presented. The main bottlenecks and weaknesses are discussed, especially regarding the analysis of morphological parameters, cell quantification and viability, gene expression profiles, metabolic behavior and high-content analysis. Finally, a vast set of applications of spheroids as tools forin vitrostudy model systems is examined, including drug screening, tissue formation, pathologies development, tissue engineering and biofabrication, 3D bioprinting and microfluidics, together with their use in high-throughput platforms.

Tuning Cell Behavior on 3D Scaffolds Fabricated by Atmospheric Plasma-Assisted Additive Manufacturing.

Three-dimensional (3D) scaffolds with optimum physicochemical properties are able to elicit specific cellular behaviors and guide tissue formation. However, cell-material interactions are limited in scaffolds fabricated by melt extrusion additive manufacturing (ME-AM) of synthetic polymers, and plasma treatment can be used to render the surface of the scaffolds more cell adhesive. In this study, a hybrid AM technology, which combines a ME-AM technique with an atmospheric pressure plasma jet, was employed to fabricate and plasma treat scaffolds in a single process. The organosilane monomer (3-aminopropyl)trimethoxysilane (APTMS) and a mixture of maleic anhydride and vinyltrimethoxysilane (MA-VTMOS) were used for the first time to plasma treat 3D scaffolds. APTMS treatment deposited plasma-polymerized films containing positively charged amine functional groups, while MA-VTMOS introduced negatively charged carboxyl groups on the 3D scaffolds' surface. Argon plasma activation was used as a control. All plasma treatments increased the surface wettability and protein adsorption to the surface of the scaffolds and improved cell distribution and proliferation. Notably, APTMS-treated scaffolds also allowed cell attachment by electrostatic interactions in the absence of serum. Interestingly, cell attachment and proliferation were not significantly affected by plasma treatment-induced aging. Also, while no significant differences were observed between plasma treatments in terms of gene expression, human mesenchymal stromal cells (hMSCs) could undergo osteogenic differentiation on aged scaffolds. This is probably because osteogenic differentiation is rather dependent on initial cell confluency and surface chemistry might play a secondary role.

3D additive manufactured composite scaffolds with antibiotic-loaded lamellar fillers for bone infection prevention and tissue regeneration.

Bone infections following open bone fracture or implant surgery remain a challenge in the orthopedics field. In order to avoid high doses of systemic drug administration, optimized local antibiotic release from scaffolds is required. 3D additive manufactured (AM) scaffolds made with biodegradable polymers are ideal to support bone healing in non-union scenarios and can be given antimicrobial properties by the incorporation of antibiotics. In this study, ciprofloxacin and gentamicin intercalated in the interlamellar spaces of magnesium aluminum layered double hydroxides (MgAl) and α-zirconium phosphates (ZrP), respectively, are dispersed within a thermoplastic polymer by melt compounding and subsequently processed via high temperature melt extrusion AM (~190 °C) into 3D scaffolds. The inorganic fillers enable a sustained antibiotics release through the polymer matrix, controlled by antibiotics counterions exchange or pH conditions. Importantly, both antibiotics retain their functionality after the manufacturing process at high temperatures, as verified by their activity against both Gram + and Gram - bacterial strains. Moreover, scaffolds loaded with filler-antibiotic do not impair human mesenchymal stromal cells osteogenic differentiation, allowing matrix mineralization and the expression of relevant osteogenic markers. Overall, these results suggest the possibility of fabricating dual functionality 3D scaffolds via high temperature melt extrusion for bone regeneration and infection prevention.

Probing the pH Microenvironment of Mesenchymal Stromal Cell Cultures on Additive-Manufactured Scaffolds.

Despite numerous advances in the field of tissue engineering and regenerative medicine, monitoring the formation of tissue regeneration and its metabolic variations during culture is still a challenge and mostly limited to bulk volumetric assays. Here, a simple method of adding capsules-based optical sensors in cell-seeded 3D scaffolds is presented and the potential of these sensors to monitor the pH changes in space and time during cell growth is demonstrated. It is shown that the pH decreased over time in the 3D scaffolds, with a more prominent decrease at the edges of the scaffolds. Moreover, the pH change is higher in 3D scaffolds compared to monolayered 2D cell cultures. The results suggest that this system, composed by capsules-based optical sensors and 3D scaffolds with predefined geometry and pore architecture network, can be a suitable platform for monitoring pH variations during 3D cell growth and tissue formation. This is particularly relevant for the investigation of 3D cellular microenvironment alterations occurring both during physiological processes, such as tissue regeneration, and pathological processes, such as cancer evolution.

Scaffold-free and label-free biofabrication technology using levitational assembly in a high magnetic field.

The feasibility of magnetic levitational bioassembly of tissue-engineered constructs from living tissue spheroids in the presence of paramagnetic ions (i.e. Gd3+) was recently demonstrated. However, Gd3+ is relatively toxic at concentrations above 50 mM normally used to enable magnetic levitation with NdFeB-permanent magnets. Using a high magnetic field (a 50 mm-bore, 31 T Bitter magnet) at the High Field Magnet Laboratory at Radboud University in Nijmegen, The Netherlands, we performed magnetic levitational assembly of tissue constructs from living spheroids prepared from the SW1353 chondrosarcoma cell line at 0.8 mM Gd3+ containing salt gadobutrol at 19 T magnetic field. The parameters of the levitation process were determined on the basis of polystyrene beads with a 170 µm-diameter. To predict the theoretical possibility of assembly, a zone of stable levitation in the horizontal and vertical areas of cross sections was previously calculated. The construct from tissue spheroids partially fused after 3 h in levitation. The analysis of viability after prolonged exposure (1 h) to strong magnetic fields (up to 30 T) showed the absence of significant cytotoxicity or morphology changes in the tissue spheroids. A high magnetic field works as a temporal and removal support or so-called 'scaffield'. Thus, formative biofabrication of tissue-engineered constructs from tissue spheroids in the high magnetic field is a promising research direction.

Dimensionality changes actin network through lamin A/C and zyxin.

Mechanosensing proteins have mainly been investigated in 2D culture platforms, while understanding their regulation in 3D enviroments is critical for tissue engineering. Among mechanosensing proteins, the actin cytoskeleton plays a key role in human mesenchymal stromal cells (hMSCs) activity, but its regulation in 3D tissue engineered scaffolds remains poorly studied. Here, we show that human mesenchymal stromal cells (hMSCs) cultured on 3D electrospun scaffolds made of a stiff material do not form actin stress fibers, contrary to hMSCs on 2D films of the same material. On 3D electrospun and additive manufactured scaffolds, hMSCs also displayed fewer focal adhesions, lower lamin A and C expression and less YAP1 nuclear localization and myosin light chain phosphorylation. Together, this strongly suggests that dimensionality prevents the build-up of cellular tension, even on stiff materials. Knock down of either lamin A and C or zyxin resulted in fewer stress fibers in the cell center. Zyxin knock down reduced lamin A and C expression, but not vice versa, showing that this signal chain starts from the outside of the cell. Lineage commitment was not affected by the lack of these important osteogenic proteins in 3D, as all cells committed to osteogenesis in bi-potential medium. Our study demonstrates that dimensionality changes the actin cytoskeleton through lamin A and C and zyxin, and highlights the difference in the regulation of lineage commitment in 3D enviroments. Together, these results can have important implications for future scaffold design for both stiff- and soft tissue engineering constructs.

Improving cell distribution on 3D additive manufactured scaffolds through engineered seeding media density and viscosity.

In order to ensure the long-term in vitro and in vivo functionality of cell-seeded 3D scaffolds, an effective and reliable method to control cell seeding efficiency and distribution is crucial. Static seeding on 3D additive manufactured scaffolds made of synthetic polymers still remains challenging, as it often results in poor cell attachment, high cell sedimentation and non-uniform cell distribution, due to gravity and to the intrinsic macroporosity and surface chemical properties of the scaffolds. In this study, the biocompatible macromolecules dextran and Ficoll (Ficoll-Paque) were used for the first time as temporary supplements to alter the viscosity and density of the seeding media, respectively, and improve the static seeding output. The addition of these macromolecules drastically reduced the cell sedimentation velocities, allowing for homogeneous cell attachment to the scaffold filaments. Both dextran and Ficoll-Paque -based seeding methods supported human mesenchymal stromal cells viability and osteogenic differentiation post-seeding. Interestingly, the improved cell distribution led to increased matrix production and mineralization compared to scaffolds seeded by conventional static method. These results suggest a simple and universal method for an efficient seeding of 3D additive manufactured scaffolds, independent of their material and geometrical properties, and applicable for bone and various other tissue regeneration. STATEMENT OF SIGNIFICANCE: Additive manufacturing has emerged as one of the desired technologies to fabricate complex and patient-specific 3D scaffolds for bone regeneration. Along with the technology, new synthetic polymeric materials have been developed to meet processability requirements, as well as the mechanical properties and biocompatibility necessary for the application. Yet, there is still lack of methodology for a universal cell seeding method applicable to all additive manufactured 3D scaffolds regardless of their characteristics. We believe that our simple and reliable method, which is based on adjusting the cell settling velocity to aid cell attachment, could potentially help to maximize the efficiency, and therefore, functionality of cell-seeded constructs. This is of great importance when aiming for both in vitro and future clinical applications.