Polyethylenimine Inclusion to Develop Aqueous Alginate-Based Core-Shell Capsules for Biomedical Applications.

Aqueous core-shell structures can serve as an efficient approach that allows cells to generate 3D spheroids with in vivo-like cell-to-cell contacts. Here, a novel strategy for fabricating liquid-core-shell capsules is proposed by inverse gelation of alginate (ALG) and layer-by-layer (LbL) coating. We hypothesized that the unique properties of polyethylenimine (PEI) could be utilized to overcome the low structural stability and the limited cell recognition motifs of ALG. In the next step, alginate dialdehyde (ADA) enabled the Schiff-base reaction with free amine groups of PEI to reduce its possible toxic effects. Scanning electron microscopy and light microscopy images proved the formation of spherical hollow capsules with outer diameters of 3.0 ± 0.1 mm for ALG, 3.2 ± 0.1 mm for ALG/PEI, and 4.0 ± 0.2 mm for ALG/PEI/ADA capsules. The effective modulus increased by 3-fold and 5-fold when comparing ALG/PEI/ADA and ALG/PEI to ALG capsules, respectively. Moreover, PEI-coated capsules showed potential antibacterial properties against both Staphylococcus aureus and Escherichia coli, with an apparent inhibition zone. The cell viability results showed that all capsules were cytocompatible (above 75.5%). Cells could proliferate and form spheroids when encapsulated within the ALG/PEI/ADA capsules. Monitoring the spheroid thickness over 5 days of incubation indicated an increasing trend from 39.50 µm after 1 day to 66.86 µm after 5 days. The proposed encapsulation protocol represents a new in vitro platform for developing 3D cell cultivation and can be adapted to fulfill the requirements of various biomedical applications.

Engineering peptide-modified alginate-based bioinks with cell-adhesive properties for biofabrication.

Alginate (ALG) and its oxidised form alginate-dialdehyde (ADA) are highly attractive materials for hydrogels used in 3D bioprinting as well as drop-on-demand (DoD) approaches. However, both polymers need to be modified using cell-adhesive peptide sequences, to obtain bioinks exhibiting promising cell-material interactions. Our study explores the modification of ALG- and ADA-based bioinks with the adhesive peptides YIGSR (derived from laminin), RRETEWA (derived from fibronectin) and IKVAV (derived from laminin) for 3D bioprinting. Two coupling methods, carbodiimide and Schiff base reactions, were employed to modify the polymers with peptides. Analytical techniques, including FTIR and NMR were used to assess the chemical composition, revealing challenges in confirming the presence of peptides. The modified bioinks exhibited decreased stability, viscosity, and stiffness, particularly-ADA-based bioinks in contrast to ALG. Sterile hydrogel capsules or droplets were produced using a manual manufacturing process and DoD printing. NIH/3T3 cell spreading analysis showed enhanced cell spreading in carbodiimide-modified ADA, Schiff base-modified ADA, and PEG-Mal-modified ADA. The carbodiimide coupling of peptides worked for ADA, however the same was not observed for ALG. Finally, a novel mixture of all used peptides was evaluated regarding synergistic effects on cell spreading which was found to be effective, showing higher aspect ratios compared to the single peptide coupled hydrogels in all cases. The study suggests potential applications of these modified bioinks in 3D bioprinting approaches and highlights the importance of peptide selection as well as their combination for improved cell-material interactions.

Mineral matrix deposition of MC3T3-E1 pre-osteoblastic cells exposed to silicocarnotite and nagelschmidtite bioceramics: In vitro comparison to hydroxyapatite.

This work presents the effect of the silicocarnotite (SC) and nagelschmidtite (Nagel) phases on in vitro osteogenesis. The known hydroxyapatite of biological origin (BHAp) was used as a standard of osteoconductive characteristics. The evaluation was carried out in conventional and osteogenic media for comparative purposes to assess the osteogenic ability of the bioceramics. First, the effect of the material on cell viability at 24 h, 7 and 14 days of incubation was evaluated. In addition, cell morphology and attachment on dense bioceramic surfaces were observed by fluorescence microscopy. Specifically, alkaline phosphatase (ALP) activity was evaluated as an osteogenic marker of the early stages of bone cell differentiation. Mineralized extracellular matrix was observed by calcium phosphate deposits and extracellular vesicle formation. Furthermore, cell phenotype determination was confirmed by scanning electron microscope. The results provided relevant information on the cell attachment, proliferation, and osteogenic differentiation processes after 7 and 14 days of incubation. Finally, it was demonstrated that SC and Nagel phases promote cell proliferation and differentiation, while the Nagel phase exhibited a superior osteoconductive behavior and could promote MC3T3-E1 cell differentiation to a higher extent than SC and BHAp, which was reflected in a higher number of deposits in a shorter period for both conventional and osteogenic media.

Amorphous, Carbonated Calcium Phosphate and Biopolymer-Composite-Coated Si3N4/MWCNTs as Potential Novel Implant Materials.

A biodegradable amorphous carbonated calcium phosphate (caCP)-incorporated polycaprolactone (PCL) composite layer was successfully deposited by a spin coater. In this specific coating, the PCL acts as a bioadhesive, since it provides a better adherence of the coatings to the substrate compared to powder coatings. The caCP-PCL coatings were deposited and formed thin layers on the surface of a Si3N4-3 wt% MWCNT (multiwalled carbon nanotube) substrate, which is an emerging type of implant material in the biomedical field. The composite coatings were examined regarding their morphology, structure and biological performance. The biocompatibility of the samples was tested in vitro with MC3T3-E1 preosteoblast cells. Owing to the caCP-PCL thin layer, the cell viability values were considerably increased compared to the substrate material. The ALP and LDH tests showed numerous living cells on the investrigated coatings. The morphology of the MC3T3-E1 cells was examined by fluorescent staining (calcein and DAPI) and scanning electron microscopy, both of which revealed a well-spread, adhered and confluent monolayer of cells. All performed biocompatibility tests were positive and indicated the applicability of the deposited thin composite layers as possible candidates for orthopaedic implants for an extended period.

On the reproducibility of extrusion-based bioprinting: round robin study on standardization in the field.

The outcome of three-dimensional (3D) bioprinting heavily depends, amongst others, on the interaction between the developed bioink, the printing process, and the printing equipment. However, if this interplay is ensured, bioprinting promises unmatched possibilities in the health care area. To pave the way for comparing newly developed biomaterials, clinical studies, and medical applications (i.e. printed organs, patient-specific tissues), there is a great need for standardization of manufacturing methods in order to enable technology transfers. Despite the importance of such standardization, there is currently a tremendous lack of empirical data that examines the reproducibility and robustness of production in more than one location at a time. In this work, we present data derived from a round robin test for extrusion-based 3D printing performance comprising 12 different academic laboratories throughout Germany and analyze the respective prints using automated image analysis (IA) in three independent academic groups. The fabrication of objects from polymer solutions was standardized as much as currently possible to allow studying the comparability of results from different laboratories. This study has led to the conclusion that current standardization conditions still leave room for the intervention of operators due to missing automation of the equipment. This affects significantly the reproducibility and comparability of bioprinting experiments in multiple laboratories. Nevertheless, automated IA proved to be a suitable methodology for quality assurance as three independently developed workflows achieved similar results. Moreover, the extracted data describing geometric features showed how the function of printers affects the quality of the printed object. A significant step toward standardization of the process was made as an infrastructure for distribution of material and methods, as well as for data transfer and storage was successfully established.

3D printing of piezoelectric and bioactive barium titanate-bioactive glass scaffolds for bone tissue engineering.

Bone healing is a complex process orchestrated by various factors, such as mechanical, chemical and electrical cues. Creating synthetic biomaterials that combine several of these factors leading to tailored and controlled tissue regeneration, is the goal of scientists worldwide. Among those factors is piezoelectricity which creates a physiological electrical microenvironment that plays an important role in stimulating bone cells and fostering bone regeneration. However, only a limited number of studies have addressed the potential of combining piezoelectric biomaterials with state-of-the-art fabrication methods to fabricate tailored scaffolds for bone tissue engineering. Here, we present an approach that takes advantage of modern additive manufacturing techniques to create macroporous biomaterial scaffolds based on a piezoelectric and bioactive ceramic-crystallised glass composite. Using binder jetting, scaffolds made of barium titanate and 45S5 bioactive glass are fabricated and extensively characterised with respect to their physical and functional properties. The 3D-printed ceramic-crystallised glass composite scaffolds show both suitable mechanical strength and bioactive behaviour, as represented by the accumulation of bone-like calcium phosphate on the surface. Piezoelectric scaffolds that mimic or even surpass bone with piezoelectric constants ranging from 1 to 21 pC/N are achieved, depending on the composition of the composite. Using MC3T3-E1 osteoblast precursor cells, the scaffolds show high cytocompatibility coupled with cell attachment and proliferation, rendering the barium titanate/45S5 ceramic-crystallised glass composites promising candidates for bone tissue engineering.

Correction: 45S5 bioactive glass-based scaffolds coated with cellulose nanowhiskers for bone tissue engineering.

[This corrects the article DOI: 10.1039/C4RA07740G.].

How to Determine a Suitable Alginate for Biofabrication Approaches using an Extensive Alginate Library?

Alginate-based hydrogels are a promising class of biomaterials due to their usability, biocompatibility, and high water-binding capacity which is the reason for their broad use in biofabrication. One challenge of these biomaterials is, however, the lack of cell adhesion motifs. This drawback can be overcome by oxidizing alginate to alginate dialdehyde (ADA) and by subsequent cross-linking with gelatin (GEL) to fabricate ADA-GEL hydrogels, which offer improved cell-material interactions. The present work investigates four pharmaceutical grade alginates of different algae sources and their respective oxidized forms regarding their molecular weight and M/G ratio using 1H NMR spectroscopy and gel permeation chromatography. In addition, three different methods for determining the degree of oxidation (% DO) of ADA, including iodometric, spectroscopic, and titration methods, are applied and compared. Furthermore, the aforementioned properties are correlated with the resulting viscosity, degradation behavior, and cell-material interactions to predict the material behavior in vitro and thus choose a suitable alginate for an intended application in biofabrication. In the framework of the present work, easy and practicable detection methods for the investigations of alginate-based bioinks were summarized and shown. In this regard, the success of oxidation of alginate was confirmed by the three aforementioned methods and was further proven by solid-state 13C NMR, for the first time in the literature, that only guluronic acid (G) was attacked during the oxidation, leading to the formation of hemiacetals. Furthermore, it was shown that ADA-GEL hydrogels of alginates with longer G-blocks are more suitable for long-term experiments due to their stability over an incubation period of 21 days, while ADA-GEL hydrogels of alginates with longer mannuronic acid (M)-blocks are more suitable for short-term applications such as sacrificial inks due to their extensive swelling and subsequent loss of shape. Finally, it was proven that the M/G ratio did not show any influence on the biocompatibility or printability of the investigated alginate-based hydrogels. The physicochemical findings provide an alginate library for tailored application in biofabrication.

Long-term stimulation with alternating electric fields modulates the differentiation and mineralization of human pre-osteoblasts.

Biophysical stimulation by electric fields can promote bone formation in bone defects of critical size. Even though, long-term effects of alternating electric fields on the differentiation of osteoblasts are not fully understood. Human pre-osteoblasts were stimulated over 31 days to gain more information about these cellular processes. An alternating electric field with 0.7 Vrms and 20 Hz at two distances was applied and viability, mineralization, gene expression, and protein release of differentiation factors were analyzed. The viability was enhanced during the first days of stimulation. A higher electric field resulted in upregulation of typical osteogenic markers like osteoprotegerin, osteopontin, and interleukin-6, but no significant changes in mineralization. Upregulation of the osteogenic markers could be detected with a lower electric field after the first days of stimulation. As a significant increase in the mineralized matrix was identified, an enhanced osteogenesis due to low alternating electric fields can be assumed.

Reinforced Hyaluronic Acid-Based Matrices Promote 3D Neuronal Network Formation.

3D neuronal cultures attempt to better replicate the in vivo environment to study neurological/neurodegenerative diseases compared to 2D models. A challenge to establish 3D neuron culture models is the low elastic modulus (30-500 Pa) of the native brain. Here, an ultra-soft matrix based on thiolated hyaluronic acid (HA-SH) reinforced with a microfiber frame is formulated and used. Hyaluronic acid represents an essential component of the brain extracellular matrix (ECM). Box-shaped frames with a microfiber spacing of 200 µm composed of 10-layers of poly(ɛ-caprolactone) (PCL) microfibers (9.7 ± 0.2 µm) made via melt electrowriting (MEW) are used to reinforce the HA-SH matrix which has an elastic modulus of 95 Pa. The neuronal viability is low in pure HA-SH matrix, however, when astrocytes are pre-seeded below this reinforced construct, they significantly support neuronal survival, network formation quantified by neurite length, and neuronal firing shown by Ca2+ imaging. The astrocyte-seeded HA-SH matrix is able to match the neuronal viability to the level of Matrigel, a gold standard matrix for neuronal culture for over two decades. Thus, this 3D MEW frame reinforced HA-SH composite with neurons and astrocytes constitutes a reliable and reproducible system to further study brain diseases.

Calcium supplementation of bioinks reduces shear stress-induced cell damage during bioprinting.

During bioprinting, cells are suspended in a viscous bioink and extruded under pressure through small diameter printing needles. The combination of high pressure and small needle diameter exposes cells to considerable shear stress, which can lead to cell damage and death. Approaches to monitor and control shear stress-induced cell damage are currently not well established. To visualize the effects of printing-induced shear stress on plasma membrane integrity, we add FM 1-43 to the bioink, a styryl dye that becomes fluorescent when bound to lipid membranes, such as the cellular plasma membrane. Upon plasma membrane disruption, the dye enters the cell and also stains intracellular membranes. Extrusion of alginate-suspended NIH/3T3 cells through a 200µm printing needle led to an increased FM 1-43 incorporation at high pressure, demonstrating that typical shear stresses during bioprinting can transiently damage the plasma membrane. Cell imaging in a microfluidic channel confirmed that FM 1-43 incorporation is caused by cell strain. Notably, high printing pressure also impaired cell survival in bioprinting experiments. Using cell types of different stiffnesses, we find that shear stress-induced cell strain, FM 1-43 incorporation and cell death were reduced in stiffer compared to softer cell types and demonstrate that cell damage and death correlate with shear stress-induced cell deformation. Importantly, supplementation of the suspension medium with physiological concentrations of CaCl2greatly reduced shear stress-induced cell damage and death but not cell deformation. As the sudden influx of calcium ions is known to induce rapid cellular vesicle exocytosis and subsequent actin polymerization in the cell cortex, we hypothesize that calcium supplementation facilitates the rapid resealing of plasma membrane damage sites. We recommend that bioinks should be routinely supplemented with physiological concentrations of calcium ions to reduce shear stress-induced cell damage and death during extrusion bioprinting.

Improved 3D Printing and Cell Biology Characterization of Inorganic-Filler Containing Alginate-Based Composites for Bone Regeneration: Particle Shape and Effective Surface Area Are the Dominant Factors for Printing Performance.

The use of organic-inorganic 3D printed composites with enhanced properties in biomedical applications continues to increase. The present study focuses on the development of 3D printed alginate-based composites incorporating inorganic fillers with different shapes (angular and round), for bone regeneration. Reactive fillers (bioactive glass 13-93 and hydroxyapatite) and non-reactive fillers (inert soda-lime glass) were investigated. Rheological studies and the characterization of various extrusion-based parameters, including material throughput, printability, shape fidelity and filament fusion, were carried out to identify the parameters dominating the printing process. It was shown that the effective surface area of the filler particle has the highest impact on the printing behavior, while the filler reactivity presents a side aspect. Composites with angular particle morphologies showed the same high resolution during the printing process, almost independent from their reactivity, while composites with comparable amounts of round filler particles lacked stackability after printing. Further, it could be shown that a higher effective surface area of the particles can circumvent the need for a higher filler content for obtaining convincing printing results. In addition, it was proven that, by changing the particle shape, the critical filler content for the obtained adequate printability can be altered. Preliminary in vitro biocompatibility investigations were carried out with the bioactive glass containing ink. The 3D printed ink, forming an interconnected porous scaffold, was analyzed regarding its biocompatibility in direct or indirect contact with the pre-osteoblast cell line MC3T3-E1. Both kinds of cell tests showed increased viability and a high rate of proliferation, with complete coverage of the 3D scaffolds' surface already after 7 d post cell-seeding.

Targeted Printing of Cells: Evaluation of ADA-PEG Bioinks for Drop on Demand Approaches.

A novel approach, in the context of bioprinting, is the targeted printing of a defined number of cells at desired positions in predefined locations, which thereby opens up new perspectives for life science engineering. One major challenge in this application is to realize the targeted printing of cells onto a gel substrate with high cell survival rates in advanced bioinks. For this purpose, different alginate-dialdehyde-polyethylene glycol (ADA-PEG) inks with different PEG modifications and chain lengths (1-8 kDa) were characterized to evaluate their application as bioinks for drop on demand (DoD) printing. The biochemical properties of the inks, printing process, NIH/3T3 fibroblast cell distribution within a droplet and shear forces during printing were analyzed. Finally, different hydrogels were evaluated as a printing substrate. By analysing different PEG chain lengths with covalently crosslinked and non-crosslinked ADA-PEG inks, it was shown that the influence of Schiff's bases on the viscosity of the corresponding materials is very low. Furthermore, it was shown that longer polymer chains resulted in less stable hydrogels, leading to fast degradation rates. Several bioinks highly exhibit biocompatibility, while the calculated nozzle shear stress increased from approx. 1.3 and 2.3 kPa. Moreover, we determined the number of cells for printed droplets depending on the initial cell concentration, which is crucially needed for targeted cell printing approaches.

Molecular Changes Induced in Melanoma by Cell Culturing in 3D Alginate Hydrogels.

Alginate hydrogels have been used as a biomaterial for 3D culturing for several years. Here, gene expression patterns in melanoma cells cultivated in 3D alginate are compared to 2D cultures. It is well-known that 2D cell culture is not resembling the complex in vivo situation well. However, the use of very intricate 3D models does not allow performing high-throughput screening and analysis is highly complex. 3D cell culture strategies in hydrogels will better mimic the in vivo situation while they maintain feasibility for large-scale analysis. As alginate is an easy-to-use material and due to its favorable properties, it is commonly applied as a bioink component in the growing field of cell encapsulation and biofabrication. Yet, only a little information about the transcriptome in 3D cultures in hydrogels like alginate is available. In this study, changes in the transcriptome based on RNA-Seq data by cultivating melanoma cells in 3D alginate are analyzed and reveal marked changes compared to cells cultured on usual 2D tissue culture plastic. Deregulated genes represent valuable cues to signaling pathways and molecules affected by the culture method. Using this as a model system for tumor cell plasticity and heterogeneity, EGR1 is determined to play an important role in melanoma progression.

Biomimetic Calcium Phosphate Coatings for Bioactivation of Titanium Implant Surfaces: Methodological Approach and In Vitro Evaluation of Biocompatibility.

Ti6Al4V as a common implant material features good mechanical properties and corrosion resistance. However, untreated, it lacks bioactivity. In contrast, coatings with calcium phosphates (CaP) were shown to improve cell-material interactions in bone tissue engineering. Therefore, this work aimed to investigate how to tailor biomimetic CaP coatings on Ti6Al4V substrates using modified biomimetic calcium phosphate (BCP) coating solutions. Furthermore, the impact of substrate immersion in a 1 M alkaline CaCl2 solution (pH = 10) on subsequent CaP coating formation was examined. CaP coatings were characterized via scanning electron microscopy, x-ray diffraction, energy-dispersive x-ray spectroscopy, and laser-scanning microscope. Biocompatibility of coatings was carried out with primary human osteoblasts analyzing cell morphology, proliferation, collagen type 1, and interleukin 6 and 8 release. Results indicate a successful formation of low crystalline hydroxyapatite (HA) on top of every sample after immersion in each BCP coating solution after 14 days. Furthermore, HA coating promoted cell proliferation and reduced the concentration of interleukins compared to the uncoated surface, assuming increased biocompatibility.

Amorphous Carbon Coatings for Total Knee Replacements-Part I: Deposition, Cytocompatibility, Chemical and Mechanical Properties.

Diamond-like carbon (DLC) coatings have the potential to reduce implant wear and thus to contribute to avoiding premature failure and increase service life of total knee replacements (TKAs). This two-part study addresses the development of such coatings for ultrahigh molecular weight polyethylene (UHMWPE) tibial inlays as well as cobalt-chromium-molybdenum (CoCr) and titanium (Ti64) alloy femoral components. While a detailed characterization of the tribological behavior is the subject of part II, part I focusses on the deposition of pure (a-C:H) and tungsten-doped hydrogen-containing amorphous carbon coatings (a-C:H:W) and the detailed characterization of their chemical, cytological, mechanical and adhesion behavior. The coatings are fabricated by physical vapor deposition (PVD) and display typical DLC morphology and composition, as verified by focused ion beam scanning electron microscopy and Raman spectroscopy. Their roughness is higher than that of the plain substrates. Initial screening with contact angle and surface tension as well as in vitro testing by indirect and direct application indicate favorable cytocompatibility. The DLC coatings feature excellent mechanical properties with a substantial enhancement of indentation hardness and elastic modulus ratios. The adhesion of the coatings as determined in modified scratch tests can be considered as sufficient for the use in TKAs.

From Thermogelling Hydrogels toward Functional Bioinks: Controlled Modification and Cytocompatible Crosslinking.

Hydrogels are key components in bioink formulations to ensure printability and stability in biofabrication. In this study, a well-known Diels-Alder two-step post-polymerization modification approach is introduced into thermogelling diblock copolymers, comprising poly(2-methyl-2-oxazoline) and thermoresponsive poly(2-n-propyl-2-oxazine). The diblock copolymers are partially hydrolyzed and subsequently modified by acid/amine coupling with furan and maleimide moieties. While the thermogelling and shear-thinning properties allow excellent printability, trigger-less cell-friendly Diels-Alder click-chemistry yields long-term shape-fidelity. The introduced platform enables easy incorporation of cell-binding moieties (RGD-peptide) for cellular interaction. The hydrogel is functionalized with RGD-peptides using thiol-maleimide chemistry and cell proliferation as well as morphology of fibroblasts seeded on top of the hydrogels confirm the cell adhesion facilitated by the peptides. Finally, bioink formulations are tested for biocompatibility by incorporating fibroblasts homogenously inside the polymer solution pre-printing. After the printing and crosslinking process good cytocompatibility is confirmed. The established bioink system combines a two-step approach by physical precursor gelation followed by an additional chemical stabilization, offering a broad versatility for further biomechanical adaptation or bioresponsive peptide modification.

An Inverse Thermogelling Bioink Based on an ABA-Type Poly(2-oxazoline) Amphiphile.

Hydrogels are key components in several biomedical research areas such as drug delivery, tissue engineering, and biofabrication. Here, a novel ABA-type triblock copolymer comprising poly(2-methyl-2-oxazoline) as the hydrophilic A blocks and poly(2-phenethyl-2-oxazoline) as the aromatic and hydrophobic B block is introduced. Above the critical micelle concentration, the polymer self-assembles into small spherical polymer micelles with a hydrodynamic radius of approx 8-8.5 nm. Interestingly, this specific combination of hydrophilic and hydrophobic aromatic moieties leads to rapid thermoresponsive inverse gelation at polymer concentrations above a critical gelation concentration (20 wt %) into a macroporous hydrogel of densely packed micelles. This hydrogel exhibited pronounced viscoelastic solid-like properties, as well as extensive shear-thinning, rapid structure recovery, and good strain resistance properties. Excellent 3D-printability of the hydrogel at lower temperature opens a wide range of different applications, for example, in the field of biofabrication. In preliminary bioprinting experiments using NIH 3T3 cells, excellent cell viabilities of more than 95% were achieved. The particularly interesting feature of this novel material is that it can be used as a printing support in hybrid bioink systems and sacrificial bioink due to rapid dissolution at physiological conditions.

Synthesis, Characterization, Antibacterial Properties, and In Vitro Studies of Selenium and Strontium Co-Substituted Hydroxyapatite.

In this study, as a measure to enhance the antimicrobial activity of biomaterials, the selenium ions have been substituted into hydroxyapatite (HA) at different concentration levels. To balance the potential cytotoxic effects of selenite ions (SeO32-) in HA, strontium (Sr2+) was co-substituted at the same concentration. Selenium and strontium-substituted hydroxyapatites (Se-Sr-HA) at equal molar ratios of x Se/(Se + P) and x Sr/(Sr + Ca) at (x = 0, 0.01, 0.03, 0.05, 0.1, and 0.2) were synthesized via the wet precipitation route and sintered at 900 °C. The effect of the two-ion concentration on morphology, surface charge, composition, antibacterial ability, and cell viability were studied. X-ray diffraction verified the phase purity and confirmed the substitution of selenium and strontium ions. Acellular in vitro bioactivity tests revealed that Se-Sr-HA was highly bioactive compared to pure HA. Se-Sr-HA samples showed excellent antibacterial activity against both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus carnosus) bacterial strains. In vitro cell-material interaction, using human osteosarcoma cells MG-63 studied by WST-8 assay, showed that Se-HA has a cytotoxic effect; however, the co-substitution of strontium in Se-HA offsets the negative impact of selenium and enhanced the biological properties of HA. Hence, the prepared samples are a suitable choice for antibacterial coatings and bone filler applications.

Neuronal Differentiation from Induced Pluripotent Stem Cell-Derived Neurospheres by the Application of Oxidized Alginate-Gelatin-Laminin Hydrogels.

Biodegradable hydrogels that promote stem cell differentiation into neurons in three dimensions (3D) are highly desired in biomedical research to study drug neurotoxicity or to yield cell-containing biomaterials for neuronal tissue repair. Here, we demonstrate that oxidized alginate-gelatin-laminin (ADA-GEL-LAM) hydrogels facilitate neuronal differentiation and growth of embedded human induced pluripotent stem cell (hiPSC) derived neurospheres. ADA-GEL and ADA-GEL-LAM hydrogels exhibiting a stiffness close to ~5 kPa at initial cell culture conditions of 37 °C were prepared. Laminin supplemented ADA-GEL promoted an increase in neuronal differentiation in comparison to pristine ADA-GEL, with enhanced neuron migration from the neurospheres to the bulk 3D hydrogel matrix. The presence of laminin in ADA-GEL led to a more than two-fold increase in the number of neurospheres with migrated neurons. Our findings suggest that laminin addition to oxidized alginate-gelatin hydrogel matrices plays a crucial role to tailor oxidized alginate-gelatin hydrogels suitable for 3D neuronal cell culture applications.