Shining a Light on Cancer-Photonics in Microfluidic Tumor Modeling and Biosensing.

Microfluidic platforms represent a powerful approach to miniaturizing important characteristics of cancers, improving in vitro testing by increasing physiological relevance. Different tools can manipulate cells and materials at the microscale, but few offer the efficiency and versatility of light and optical technologies. Moreover, light-driven technologies englobe a broad toolbox for quantifying critical biological phenomena. Herein, the role of photonics in microfluidic 3D cancer modeling and biosensing from three major perspectives is reviewed. First, optical-driven technologies are looked upon, as these allow biomaterials and living cells to be manipulated with microsized precision and present opportunities to advance 3D microfluidic models by engineering cancer microenvironments' hallmarks, such as their architecture, cellular complexity, and vascularization. Second, the growing field of optofluidics is discussed, exploring how optical tools can directly interface microfluidic chips, enabling the extraction of relevant biological data, from single fluorescent signals to the complete 3D imaging of diseased cells within microchannels. Third, advances in optical cancer biosensing are reviewed, focusing on how light-matter interactions can detect biomarkers, rare circulating tumor cells, and cell-derived structures such as exosomes. Photonic technologies' current challenges and caveats in microfluidic 3D cancer models are overviewed, outlining future research avenues that may catapult the field.

Dataset of analyzes performed to determine the level and timing of selected organic pollutants' inputs in sediments of the Lake of Cavazzo (Italy).

This data article presents the dataset collected for selected organic pollutants in the framework of a larger research project aimed at assessing the effects of different environmental stressors (natural and anthropogenic) in sediments of the Lake of Cavazzo, a basin of glacial origin located in a seismically active region of the Italian Eastern Alps. Information relative to sampling strategy and operations, location of sampling sites, sedimentary chronological benchmarks, and profiles of RGB (Red-Green-Blue) color code determined from high resolution photos taken at cores CAV-04 and CAV-06 are reported, together with analytical data for 15 polycyclic aromatic hydrocarbons, 21 polychlorinated biphenyls' congeners (including the non-Aroclor CB-11), 14 polybrominated diphenyl ethers' congeners, and 22 organochlorine pesticides, whose concentrations were determined by Gas Chromatography coupled both to Low-Resolution and High-Resolution Mass Spectrometry. Interpretation of this dataset is fully discussed in the companion article by Pizzini et al. (2022) and relys on the multi-proxy analysis of sediment samples presented in Polonia et al. (2021) that highlighted lake stratigraphy and major changes occurring at a decadal scale since the 1950s.

PAHs, PCBs, PBDEs, and OCPs trapped and remobilized in the Lake of Cavazzo (NE Italy) sediments: Temporal trends, quality, and sources in an area prone to anthropogenic and natural stressors.

Under the present climatic emergency, the environmental quality of freshwater reservoirs is an increasingly urgent topic as its deterioration threatens humans and ecosystems. It is evident that pollution by natural and anthropogenic contaminants must be avoided or reduced. The Lake of Cavazzo (NE Italy) is a natural perialpine basin which, from the mid-20th century, has sustained several anthropogenic impacts that added to the effects of the intense regional seismicity. Starting from 2015, in response to concerns raised by local authorities, a multidisciplinary investigation of the lake floor and sub-floor was conducted, including a geophysical survey and the collection of sediment cores. Two of them were studied to detect contamination by Polycyclic Aromatic Hydrocarbons (PAHs) and specific Persistent Organic Pollutants (POPs; i.e. PolyChlorinated Biphenyls - PCBs, PolyBrominated Diphenyl Ethers - PBDEs, and OrganoChlorine Pesticides - OCPs), and to verify the link with known anthropogenic stressors. Results were interpreted in light of previous studies suggesting modified conditions after the '50s, and recognized the effects of the 1976-1977 MW 6.5 seismic sequence in resuspending sediments within the basin. Analyzed pollutants defined a potential critical situation only for few OCPs, above all 2,4'- and 4,4'-DDT isomers. In addition, PBDEs were found at concentrations exceeding those of other heavily polluted alpine lakes. Mass movements (either seismic or human induced) have likely resuspended and transferred pollutants from shallower locations to the lake depocenter, showing the potential of re-exposing contaminated layers to biomagnification processes along the lacustrine food chain. Local inputs of pollutants prevail over distributed sources, suggesting a link with local agricultural or industrial activities. Indeed, works connected to the construction of the hydroelectric power plant in the '50s might have reworked local sediments perturbing their natural accretion. Results of this work might inspire similar studies in other problematic lacustrine areas that sustain both natural and anthropogenic stressors.

The International Bathymetric Chart of the Southern Ocean Version 2.

The Southern Ocean surrounding Antarctica is a region that is key to a range of climatic and oceanographic processes with worldwide effects, and is characterised by high biological productivity and biodiversity. Since 2013, the International Bathymetric Chart of the Southern Ocean (IBCSO) has represented the most comprehensive compilation of bathymetry for the Southern Ocean south of 60°S. Recently, the IBCSO Project has combined its efforts with the Nippon Foundation - GEBCO Seabed 2030 Project supporting the goal of mapping the world's oceans by 2030. New datasets initiated a second version of IBCSO (IBCSO v2). This version extends to 50°S (covering approximately 2.4 times the area of seafloor of the previous version) including the gateways of the Antarctic Circumpolar Current and the Antarctic circumpolar frontal systems. Due to increased (multibeam) data coverage, IBCSO v2 significantly improves the overall representation of the Southern Ocean seafloor and resolves many submarine landforms in more detail. This makes IBCSO v2 the most authoritative seafloor map of the area south of 50°S.

Microfluidic-Driven Biofabrication and the Engineering of Cancer-Like Microenvironments.

Despite considerable advances in cancer research and oncological treatments, the burden of the disease is still extremely high. While past research has been cancer cell centered, it is now clear that to understand tumors, the models that serve as a framework for research and therapeutic testing need to improve and integrate cancer microenvironment characteristics such as mechanics, architecture, and cell heterogeneity. Microfluidics is a powerful tool for biofabrication of cancer-relevant architectures given its capacity to manipulate cells and materials at very small dimensions and integrate varied living tissue characteristics. This chapter outlines the current microfluidic toolbox for fabricating living constructs, starting by explaining the varied configurations of 3D soft constructs microfluidics enables when used to process hydrogels. Then, we analyze the possibilities to control material flows and create space varying characteristics such as gradients or advanced 3D micro-architectures. Envisioning the trend to approach the complexity of tumor microenvironments also at higher dimensions, we discuss microfluidic-enabled 3D bioprinting and recent advances in that arena. Finally, we summarize the future possibilities for microfluidic biofabrication to tackle important challenges in cancer 3D modelling, including tools for the fast quantification of biological events toward data-driven and precision medicine approaches.

Bioinks Enriched with ECM Components Obtained by Supercritical Extraction.

Extracellular matrix (ECM)-based bioinks have been steadily gaining interest in the field of bioprinting to develop biologically relevant and functional tissue constructs. Herein, we propose the use of supercritical carbon dioxide (scCO2) technology to extract the ECM components of cell-sheets that have shown promising results in creating accurate 3D microenvironments replicating the cell's own ECM, to be used in the preparation of bioinks. The ECM extraction protocol best fitted for cell sheets was defined by considering efficient DNA removal with a minor effect on the ECM. Cell sheets of human dermal fibroblasts (hDFbs) and adipose stem cells (hASCs) were processed using a customised supercritical system by varying the pressure of the reactor, presence, exposure time, and type of co-solvent. A quantification of the amount of DNA, protein, and sulfated glycosaminoglycans (sGAGs) was carried out to determine the efficiency of the extraction in relation to standard decellularization methodologies. The bioinks containing the extracted ECM were fabricated by combining them with alginate as a support polymer. The influence of the alginate (1%, 2% w/vol) and ECM (0.5% and 1.5% w/vol) amounts on the printability of the blends was addressed by analysing the rheological behaviour of the suspensions. Finally, 3D printed constructs were fabricated using an in-house built extrusion-based bioprinter, and the impact of the extrusion process on cell viability was assessed. The optimised scCO2 protocol allowed efficient removal of DNA while preserving a higher number of proteins and sGAGs than the standard methodologies. The characterization of extract's composition also revealed that the ECM produced by hDFbs (fECM) and hASCs (aECM) is distinctively affected by the extraction protocols. Furthermore, rheological analysis indicated an increase in viscosity with increasing ECM composition, an effect even more prominent in samples containing aECM. 3D printing of alginate/ECM constructs demonstrated that cell viability was only marginally affected by the extrusion process, and this effect was also dependent on the ECM source. Overall, this work highlights the benefits of supercritical fluid-based methods for ECM extraction and strengthens the relevance of ECM-derived bioinks in the development of printed tissue-like constructs.

Numerical and experimental simulation of a dynamic-rotational 3D cell culture for stratified living tissue models.

Human tissues and organs are inherently heterogeneous, and their functionality is determined by the interplay between different cell types, their secondary architecture, and gradients of signalling molecules and metabolites. To mimic the dynamics of native tissues, perfusion bioreactors and microfluidic devices are widely used in tissue engineering (TE) applications for enhancing cell culture viability in the core of 3D constructs. Still, mostin vitroscreening methods for compound efficacy and toxicity assessment include cell or tissue exposure to constant and homogeneous compound concentrations over a defined testing period. Moreover, a prevalent issue inhibiting the large-scale adoption of microfluidics and bioreactor is the tubing dependence to induce a perfusion regime. Here, we propose a compartmentalized rotational (CR) 3D cell culture platform for a stable control over gradient tissue culture conditions. Using the CR bioreactor, adjacent lanes of constructs are patterned by controlled flow dynamics to enable tissue stratification. Numerical and experimental simulations demonstrate cell seeding dynamics, as well as culture media rotational perfusion and gradient formations. Additionally, the developed system induces vertical and horizontal rotations, which increase medium exchange and homogeneous construct maturation, allowing both perfused tubing-based and tubing-free approaches. As a proof-of-concept, experiments and accompanying simulation of cellular inoculation and growth in 3D scaffold and hydrogel were performed, before the examination of a blood-brain-barrier model, demonstrating the impact of a heterotypic culture on molecular permeability under mimetic dynamic conditions. Briefly, the present work discloses the simulation of 3D dynamic cultures, and a semi-automated platform for heterotypic tissuesin vitromodelling, for broad TE and drug discovery/screening applications.

Multidisciplinary dataset for geological and environmental studies in the lake of Cavazzo (Southern Alps).

The present dataset was collected to evaluate the environmental stressors on a lacustrine basin in the Eastern Alps of glacial origin that has been affected in recent years by natural and anthropogenic events such as the construction of a hydroelectric power plant and a series of strong earthquakes during 1976-1977. We collected sediment cores in different sites from the lake margins to the depocenter and performed a multiproxy analysis of sediment sample to highlight lake stratigraphy and major changes occurring at a decadal scale (Polonia et al., [1]). The integrated analyses of sedimentological, geochemical, isotopic, mineralogical and micropaleontological analyses aimed at reconstructing changes in sediment composition and define the triggering mechanisms of altered environmental conditions. The dataset demonstrates that evaluating ex post the effects of artificial modification in a natural environment during relatively long time spans (decades) can provide important insights for managing and protection strategies in similar environments worldwide.

Rescuing key native traits in cultured dermal papilla cells for human hair regeneration.

Introduction: The dermal papilla (DP) represents the major regulatory entity within the hair follicle (HF), inducing hair formation and growth through reciprocal interactions with epithelial cells. However, human DP cells rapidly lose their hair inductive ability when cultured in an epithelium-deficient environment. Objectives: To determine if the conditioned medium collected from interfollicular keratinocytes (KCs-CM) is capable of improving DP cell native properties and inductive phenotype. Methods: DP cells were cultured with KCs-CM both in 2D and 3D culture conditions (spheroids). Further, the hair-inductive capacity of DP cells precultured with KCs-CM was tested in a hair reconstitution assay, after co-grafting with human keratinocytes in nude mice. Results: We demonstrate that KCs-CM contributes to restore the inductivity of cultured human DP cells in a more effective mode than the conventional 3D-cultures. This is supported by the higher active alkaline phosphatase (ALP) levels in DP cells, the improved self-aggregative capacity and the reduced expression of α-SMA and the V1-isoform of versican. Moreover, DP cells cultured with KCs-CM displayed a secretome profile (VEGF, BMP2, TGF- ß1, IL-6) that matches the one observed during anagen. KCs-CM also enhanced DP cell proliferation, while preventing cells to undergo morphological changes characteristic of high passage cells. In opposition, the amount of collagenous and non-collagenous proteins deposited by DP cells was lower in the presence of KCs-CM. The improvement in ALP activity was maintained in 3D spheroidal cultures, even after KCs-CM retrieval, being superior to the effect of the gold-standard culture conditions. Moreover, DP cells cultured with KCs-CM and grafted with human keratinocytes supported the formation of HF- and sebaceous gland-like structures in mice. Conclusion: The proposed strategy encourages future cell-based strategies for HF regeneration not only in the context of hair-associated disorders, but also in the management of wounds to aid in restoring critical skin regulatory appendages.

Microscopy-guided laser ablation for the creation of complex skin models with folliculoid appendages.

Engineering complex tissues requires the use of advanced biofabrication techniques that allow the replication of the tissue's 3D microenvironment, architecture and cellular interactions. In the case of skin, the most successful strategies to introduce the complexity of hair follicle (HF) appendages have highlighted the importance of facilitating direct interaction between dermal papilla (DP) cells and keratinocytes (KCs) in organotypic skin models. In this work, we took advantage of microscopy-guided laser ablation (MGLA) to microfabricate a fibroblast-populated collagen hydrogel and create a subcompartment that guides the migration of KCs and lead their interaction with DP cells to recreate follicular structures. Upon definition of the processing parameters (laser incidence area and power), MGLA was used to create 3D microchannels from the surface of a standard organotypic human skin model up to the aggregates containing DP cells and KCs, previously incorporated into the dermal-like fibroblast-collagen layer. Analysis of the constructs showed that the fabricated microfeatures successfully guided the fusion between epidermal and aggregates keratinocytes, which differentiated into follicular-like structures within the organotypic human skin model, increasing its functionality. In summary, we demonstrate the fabrication of a highly structured 3D hydrogel-based construct using MGLA to attain a complex skin model bearing folliculoid structures, highlighting its potential use as an in vitro platform to study the mechanisms controlling HF development or for the screening of bioactive substances.

New seismological data from the Calabrian arc reveal arc-orthogonal extension across the subduction zone.

The Calabrian Arc subduction-rollback system along the convergent Africa/Eurasia plate boundary is among the most active geological structures in the Mediterranean Sea. However, its seismogenic behaviour is largely unknown, mostly due to the lack of seismological observations. We studied low-to-moderate magnitude earthquakes recorded by the seismic network onshore, integrated by data from a seafloor observatory (NEMO-SN1), to compute a lithospheric velocity model for the western Ionian Sea, and relocate seismic events along major tectonic structures. Spatial changes in the depth distribution of earthquakes highlight a major lithospheric boundary constituted by the Ionian Fault, which separates two sectors where thickness of the seismogenic layer varies over 40 km. This regional tectonic boundary represents the eastern limit of a domain characterized by thinner lithosphere, arc-orthogonal extension, and transtensional tectonic deformation. Occurrence of a few thrust-type earthquakes in the accretionary wedge may suggest a locked subduction interface in a complex tectonic setting, which involves the interplay between arc-orthogonal extension and plate convergence. We finally note that distribution of earthquakes and associated extensional deformation in the Messina Straits region could be explained by right-lateral displacement along the Ionian Fault. This observation could shed new light on proposed mechanisms for the 1908 Messina earthquake.

Crustal deformation, active tectonics and seismic potential in the Sicily Channel (Central Mediterranean), along the Nubia-Eurasia plate boundary.

Based on multidisciplinary data, including seismological and geodetic observations, as well as seismic reflection profiles and gravity maps, we analysed the pattern of crustal deformation and active tectonics in the Sicily Channel, a key observation point to unravel the complex interaction between two major plates, Nubia and Eurasia, in the Mediterranean Sea. Our data highlight the presence of an active ~ 220-km-long complex lithospheric fault system (here named the Lampedusa-Sciacca Shear Zone), approximately oriented N-S, crossing the study area with left-lateral strike-slip deformations, active volcanism and high heat flow. We suggest that this shear zone represents the most active tectonic domain in the area, while the NW-SE elongated rifting pattern, considered the first order tectonic feature, appears currently inactive and sealed by undeformed recent (Lower Pleistocene?) deposits. Estimates of seismological and geodetic moment-rates, 6.58 × 1015 Nm/year and 7.24 × 1017 Nm/year, respectively, suggests that seismicity accounts only for ~ 0.9% of crustal deformation, while the anomalous thermal state and the low thickness of the crust would significantly inhibit frictional sliding in favour of creeping and aseismic deformation. We therefore conclude that a significant amount of the estimated crustal deformation-rate occurs aseismically, opening new scenarios for seismic risk assessments in the region.

Neotectonics of the Sea of Galilee (northeast Israel): implication for geodynamics and seismicity along the Dead Sea Fault system.

The Sea of Galilee in northeast Israel is a freshwater lake filling a morphological depression along the Dead Sea Fault. It is located in a tectonically complex area, where a N-S main fault system intersects secondary fault patterns non-univocally interpreted by previous reconstructions. A set of multiscale geophysical, geochemical and seismological data, reprocessed or newly collected, was analysed to unravel the interplay between shallow tectonic deformations and geodynamic processes. The result is a neotectonic map highlighting major seismogenic faults in a key region at the boundary between the Africa/Sinai and Arabian plates. Most active seismogenic displacement occurs along NNW-SSE oriented transtensional faults. This results in a left-lateral bifurcation of the Dead Sea Fault forming a rhomb-shaped depression we named the Capharnaum Trough, located off-track relative to the alleged principal deformation zone. Low-magnitude (ML = 3-4) epicentres accurately located during a recent seismic sequence are aligned along this feature, whose activity, depth and regional importance is supported by geophysical and geochemical evidence. This case study, involving a multiscale/multidisciplinary approach, may serve as a reference for similar geodynamic settings in the world, where unravelling geometric and kinematic complexities is challenging but fundamental for reliable earthquake hazard assessments.

Microfluidics for Processing of Biomaterials.

Microfluidics techniques can be used to process a wide range of biomaterials, from synthetic to natural origin ones. This chapter describes microfluidic processing of biomaterials, mainly polymeric materials of natural origin, focusing on water-soluble polymers that form non-flowing phases after crosslinking. Some polysaccharides and proteins, including agarose, alginate, chitosan, gellan gum, hyaluronic acid, collagen, gelatin, and silk fibroin are emphasized deu to their relevance in the field. The critical characteristics of these materials are discussed, giving particular consideration to those that directly impact its processability using microfluidics. Furthermore, some microfluidic-based processing techniques are presented, describing their suitability to process materials with different sol-gel transition mechanisms.

3D Bioprinting Technology: Scientific Aspects and Ethical Issues.

The scientific development of 3D bioprinting is rapidly advancing. This innovative technology involves many ethical and regulatory issues, including theoretical, source, transplantation and enhancement, animal welfare, economic, safety and information arguments. 3D bioprinting technology requires an adequate bioethical debate in order to develop regulations in the interest both of public health and the development of research. This paper aims to initiate and promote ethical debate. The authors examine scientific aspects of 3D bioprinting technology and explore related ethical issues, with special regard to the protection of individual rights and transparency of research. In common with all new biotechnologies, 3D bioprinting technology involves both opportunities and risks. Consequently, several scientific and ethical issues need to be addressed. A bioethical debate should be carefully increased through a multidisciplinary approach among experts and also among the public.

Lake Afrera, a structural depression in the Northern Afar Rift (Red Sea).

The boundary between the African and Arabian plates in the Southern Red Sea region is displaced inland in the northern Afar rift, where it is marked by the Red Sea-parallel Erta Ale, Alaita, and Tat Ali volcanic ridges. The Erta Ale is offset by about 20 and 40 km from the two en echelon ridges to the south. The offset area is highly seismic and marked by a depression filled by lake Afrera, a saline body of water fed by hydrothermal springs. Acoustic bathymetric profiles show ≈80 m deep canyons parallel to the NNW shore of the lake, part of a system of extensional normal faults striking parallel to the Red Sea. This system is intersected by oblique structures, some with strike-slip earthquakes, in what might evolve into a transform boundary. Given that the lake's surface lies today about 112 m below sea level, the depressed (minus ≈190 m below sea level) lake's bottom area may be considered the equivalent of the "nodal deep" in slow-slip oceanic transforms. The chemistry of the lake is compatible with the water having originated from hydrothermal liquids that had reacted with evaporites and basalts, rather than residual from evaporation of sea water. Bottom sediments include calcitic grains, halite and gypsum, as well as ostracod and diatom tests. The lake's level appears to have dropped by over 10 m during the last ≈50 years, continuing a drying up trend of the last few thousand years, after a "wet" stage 9,800 and 7,800 years before present when according to Gasse (1973) Lake Afrera covered an area several times larger than at present. This "wet" stage corresponds to an early Holocene warm-humid climate that prevailed in Saharan and Sub Saharan Africa. Lake Abhé, located roughly 250 km south of Afrera, shows similar climate-driven oscillations of its level.

Synthesis, mechanical and thermal rheological properties of new gellan gum derivatives.

New derivatives of gellan gum (GG) were prepared by covalent attachment of octadecylamine (C18-NH2) to polysaccharide backbone via amide linkage by using bis(4-nitrophenyl) carbonate (4-NPBC) as a coupling agent. The effect of the alkyl chain grafted onto hydrophilic backbone of high molecular weight GG was investigated in terms of physicochemical properties and ability of new derivatives to form hydrogels. A series of hydrogels was obtained in solutions with different kind and concentration of ions and their stability and mechanical properties were evaluated. The obtained derivatives resulted soluble at temperature lower than starting GG and physicochemical properties of obtained hydrogels suggested their potential use in biomedical field.

Microengineered Multicomponent Hydrogel Fibers: Combining Polyelectrolyte Complexation and Microfluidics.

Fiber-based techniques hold great potential toward the development of structures that mimic the architecture of fibrous tissues, such as tendon. Microfluidics and polyelectrolyte complexation are among the most widely used techniques for the fabrication of fibrous structures. In this work, we combined both techniques to generate hydrogel fibers with a fibrillar-like structure. For this, either methacrylated hyaluronic acid (MA-HA) or chondroitin sulfate (MA-CS) were mixed with alginate (ALG), being all negatively charged polysaccharides, combined with chitosan (CHT), which is positively charged, and separately injected into a microfluidic device. Through a continuous injection into a coagulation bath and subsequent photo-cross-linking, we could obtain multicomponent hydrogel fibers, which exhibited smaller fibrils aligned in parallel, whenever CHT was present. The biological performance was assessed upon encapsulation and further culture of tendon cells. Overall, the reported process did not affect cell viability and cells were also able to maintain their main function of producing extracellular matrix up to 21 days in culture. In summary, we developed a novel class of photo-cross-linkable multicomponent hydrogel fibers than can act as bioactive modulators of cell behavior.

Autonomous osteogenic differentiation of hASCs encapsulated in methacrylated gellan-gum hydrogels.

UNLABELLED: Methacrylated gellan-gum (GG-MA) alone and combined with collagen type I (Coll) is suggested here for the first time as a cell-laden injectable biomaterial for bone regeneration. On-chip high-throughput studies allowed rapidly assessing the suitability of 15 biomaterials/media combinations for the osteodifferentiation of human adipose stem cells (hASCs). Hydrogels composed solely of GG-MA (GG100:0Coll) led hASCs from three different donors into the osteogenic lineage after 21days of cell culture, in the absence of any osteogenic or osteoconductive factors. Hydrogels containing more than 30% of Coll promoted increased cellular proliferation and led hASCs into osteogenic differentiation under basal conditions. Studies using isolated individual hydrogels - excluding eventual on-chip crosstalk - and standard biochemical assays corroborated such findings. The formation of focal adhesions of hASCs on GG100:0Coll hydrogels was verified. We hypothesize that the hydrogels osteogenic effect could be guided by mechanotransduction phenomena. Indeed, the hydrogels showed elastic modulus in ranges previously reported as osteoinductive and the inhibition of the actin-myosin contractility pathway impaired hASCs' osteodifferentiation. GG-MA hydrogels also did not promote hASCs' adipogenesis while used in basal conditions. Overall, GG-MA showed promising properties as an innovative and off-the shelf self-inducing osteogenic injectable biomaterial. STATEMENT OF SIGNIFICANCE: Methacrylated gellan gum (GG-MA) is here suggested for the first time as a widely available polysaccharide to easily prepare hydrogels with cell adhesion properties and capability of inducing the autonomous osteogenic differentiation of human adipose-derived stem cells (hASCs). GG-MA was processed as stand-alone hydrogels or in different combinations with collage type I. All hydrogel formulations elicited the osteogenic differentiation of hASCs, independently of the addition of any osteoconductive or osteogenic stimuli, i.e. in basal/growth medium. Effective cellular adhesion to methacrylated gellan gum hydrogels in the absence of any cell-ligand peptide/protein was here proved for the first time. Moreover, we showed that the encapsulated hASCs underwent osteogenic differentiation due to a mechanotransduction phenomenon dependent on the actin-myosin contractility pathway.

Assessing the impact of electrohydrodynamic jetting on encapsulated cell viability, proliferation, and ability to self-assemble in three-dimensional structures.

In this article, we propose a systemic approach to investigate the impact of electrohydrodynamic jetting (EHDJ) encapsulation on viability, proliferation, and functionality of the encapsulated cells. EHDJ consists in applying a high-voltage electrical field between a target substrate and a jetting needle, which is fed with a suspension of cells in a polymeric solution undergoing a sol-gel transition upon contact with the target. The viability, proliferation, and self-assembling ability of SHSY5Y human neuroblastoma cell line encapsulated in 2% alginate microbeads were analyzed by confocal microscopy and DNA quantification assays. In addition, the expression of stress (HSP70B'), apoptotic (CASP3), necrotic (HMGB1), hypoxic (HYOU1, GAPDH), and adhesion (CDH2) markers was measured with reverse transcription quantitative polymerase chain reaction (qPCR). After an initial upregulation of the HSP70B' expression within 24 h, its expression decreased to the negative control level together with a decrease in the expression of CASP3. Any increase in necrotic or hypoxic marker expression was not detected, while a slight upregulation of CDH2 was observed in the first days after encapsulation, followed by its downregulation and stabilization to the control level. Furthermore, cell-laden beads started to self-assemble in three-dimensional (3D) constructs from the 3rd week after encapsulation. The results indicated that the EHDJ encapsulation method had a mild effect on cells, which after a week, fully recovered their proliferation rate and ability to self-assemble into 3D constructs.