Gellan gum-gelatin based cardiac models support formation of cellular networks and functional cardiomyocytes.

Cardiovascular diseases remain as the most common cause of death worldwide. To reveal the underlying mechanisms in varying cardiovascular diseases, in vitro models with cells and supportive biomaterial can be designed to recapitulate the essential components of human heart. In this study, we analyzed whether 3D co-culture of cardiomyocytes (CM) with vascular network and with adipose tissue-derived mesenchymal stem/stromal cells (ASC) can support CM functionality. CM were cultured with either endothelial cells (EC) and ASC or with only ASC in hydrazide-modified gelatin and oxidized gellan gum hybrid hydrogel to form cardiovascular multiculture and myocardial co-culture, respectively. We studied functional characteristics of CM in two different cellular set-ups and analyzed vascular network formation, cellular morphology and orientation. The results showed that gellan gum-gelatin hydrogel supports formation of two different cellular networks and functional CM. We detected formation of a modest vascular network in cardiovascular multiculture and extensive ASC-derived alpha smooth muscle actin -positive cellular network in multi- and co-culture. iPSC-CM showed elongated morphology, partly aligned orientation with the formed networks and presented normal calcium transients, beating rates, and contraction and relaxation behavior in both setups. These 3D cardiac models provide promising platforms to study (patho) physiological mechanisms of cardiovascular diseases. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-024-00630-5.

Interpretable Machine Learning Methods for Monitoring Polymer Degradation in Extrusion of Polylactic Acid.

This work investigates real-time monitoring of extrusion-induced degradation in different grades of PLA across a range of process conditions and machine set-ups. Data on machine settings together with in-process sensor data, including temperature, pressure, and near-infrared (NIR) spectra, are used as inputs to predict the molecular weight and mechanical properties of the product. Many soft sensor approaches based on complex spectral data are essentially 'black-box' in nature, which can limit industrial acceptability. Hence, the focus here is on identifying an optimal approach to developing interpretable models while achieving high predictive accuracy and robustness across different process settings. The performance of a Recursive Feature Elimination (RFE) approach was compared to more common dimension reduction and regression approaches including Partial Least Squares (PLS), iterative PLS (i-PLS), Principal Component Regression (PCR), ridge regression, Least Absolute Shrinkage and Selection Operator (LASSO), and Random Forest (RF). It is shown that for medical-grade PLA processed under moisture-controlled conditions, accurate prediction of molecular weight is possible over a wide range of process conditions and different machine settings (different nozzle types for downstream fibre spinning) with an RFE-RF algorithm. Similarly, for the prediction of yield stress, RFE-RF achieved excellent predictive performance, outperforming the other approaches in terms of simplicity, interpretability, and accuracy. The features selected by the RFE model provide important insights to the process. It was found that change in molecular weight was not an important factor affecting the mechanical properties of the PLA, which is primarily related to the pressure and temperature at the latter stages of the extrusion process. The temperature at the extruder exit was also the most important predictor of degradation of the polymer molecular weight, highlighting the importance of accurate melt temperature control in the process. RFE not only outperforms more established methods as a soft sensor method, but also has significant advantages in terms of computational efficiency, simplicity, and interpretability. RFE-based soft sensors are promising for better quality control in processing thermally sensitive polymers such as PLA, in particular demonstrating for the first time the ability to monitor molecular weight degradation during processing across various machine settings.

pH-Responsive Gallol-Functionalized Hyaluronic Acid-Based Tissue Adhesive Hydrogels for Injection and Three-Dimensional Bioprinting.

The major challenges of hyaluronic acid-based bioinks in extrusion-based three-dimensional bioprinting are poor printability and low printing accuracy. To tackle the challenges, we developed a bioink in which two components are blended: gallic acid-functionalized hyaluronic acid (HAGA) and hyaluronic acid methacrylate (HAMA). In the precursor phase, the blend's HAGA component enables pH-dependent viscosity modulation that results in improved injectability and printability at physiological temperature. Postprinting, the blend's HAMA component is photocrosslinked to create a true hydrogel with a complementary network of both HAGA and HAMA. The ready structures of the HAGA-HAMA hydrogel showed sufficient printing quality and accuracy compared to plain HAMA. The blend also displayed enhanced viscoelastic properties and stable swelling behavior. In addition to the pH tunability, the HAGA component also imparted tissue adhesion and antioxidant activity. This bioink has the potential to be printed directly on an infected wound site due to its adhesiveness to tissue and dimensional stability in situ.

In vitro biocompatibility of polylactide and polybutylene succinate blends for urethral tissue engineering.

Surgical treatment of urothelial defects with autologous genital or extragenital tissue grafts is susceptible to complications. Tissue engineering utilizing novel biomaterials and cells such as human urothelial cells (hUC) for epithelial regeneration and adipose stromal cells (hASC) for smooth muscle restoration might offer new treatment options for urothelial defects. Previously, polylactide (PLA) has been studied for urethral tissue engineering, however, as such, it is too stiff and rigid for the application. Blending it with ductile polybutylene succinate (PBSu) could provide suitable mechanical properties for the application. Our aim was to study the morphology, viability and proliferation of hUC and hASC when cultured on 100/0 PLA/PBSu, 75/25 PLA/PBSu blend, 50/50 PLA/PBSu blend, and 0/100 PLA/PBSu discs. The results showed that the hUCs were viable and proliferated on all the studied materials. The hUCs stained pancytokeratin at 7 and 14 days, suggesting maintenance of the urothelial phenotype. The hASCs retained their viability and morphology and proliferated on all the other discs, except on PLA. On the PLA, the hASCs formed large aggregates with each other rather than attached to the material. The early smooth muscle cell markers SM22α and α-SMA were stained in hASC at 7 and 14 day time points on all PBSu-containing materials, indicating that hASCs maintain their smooth muscle differentiation potential also on PBSu. As a conclusion, PBSu is a highly potential biomaterial for urothelial tissue engineering since it supports growth and phenotypic maintenance of hUC and smooth muscle differentiation of hASC.

Sequential Cross-linking of Gallic Acid-Functionalized GelMA-Based Bioinks with Enhanced Printability for Extrusion-Based 3D Bioprinting.

The printability of a photocross-linkable methacrylated gelatin (GelMA) bioink with an extrusion-based 3D bioprinter is highly affected by the polymer concentration and printing temperature. In this work, we developed a gallic acid (GA)-functionalized GelMA ink to improve the printability at room and physiological temperatures and to enable tissue adhesion and antioxidant properties. We introduced a sequential cross-linking approach using catechol-Fe3+ chelation, followed by photocross-linking. The results show that the ink formulation with 0.5% (w/v) Fe3+ in GelMA (30% modification) with 10% GA (GelMA30GA-5Fe) provided the optimum printability, shape fidelity, and structural integrity. The dual network inside the printed constructs significantly enhanced the viscoelastic properties. Printed cylinders were evaluated for their printing accuracy. The printed structures of GelMA30GA-5Fe provided high stability in physiological conditions over a month. In addition, the optimized ink also offered good tissue adhesion and antioxidant property. This catechol-based sequential cross-linking method could be adopted for the fabrication of other single-polymer bioinks.

Bioactivated gellan gum hydrogels affect cellular rearrangement and cell response in vascular co-culture and subcutaneous implant models.

Hydrogels are suitable soft tissue mimics and capable of creating pre-vascularized tissues, that are useful for in vitro tissue engineering and in vivo regenerative medicine. The polysaccharide gellan gum (GG) offers an intriguing matrix material but requires bioactivation in order to support cell attachment and transfer of biomechanical cues. Here, four versatile modifications were investigated: Purified NaGG; avidin-modified NaGG combined with biotinylated fibronectin (NaGG-avd); oxidized GG (GGox) covalently modified with carbohydrazide-modified gelatin (gelaCDH) or adipic hydrazide-modified gelatin (gelaADH). All materials were subjected to rheological analysis to assess their viscoelastic properties, using a time sweep for gelation analysis, and subsequent amplitude sweep of the formed hydrogels. The sweeps show that NaGG and NaGG-avd are rather brittle, while gelatin-based hydrogels are more elastic. The degradation of preformed hydrogels in cell culture medium was analyzed with an amplitude sweep and show that gelatin-containing hydrogels degrade more dramatically. A co-culture of GFP-tagged HUVEC and hASC was performed to induce vascular network formation in 3D for up to 14 days. Immunofluorescence staining of the αSMA+ network showed increased cell response to gelatin-GG networks, while the NaGG-based hydrogels did not allow for the elongation of cells. Preformed, 3D hydrogels disks were implanted to subcutaneous rat skin pockets to evaluate biological in vivo response. As visible from the hematoxylin and eosin-stained tissue slices, all materials are biocompatible, however gelatin-GG hydrogels produced a stronger host response. This work indicates, that besides the biochemical cues added to the GG hydrogels, also their viscoelasticity greatly influences the biological response.

Chemical modification strategies for viscosity-dependent processing of gellan gum.

Recently, the hydrogel-forming polysaccharide gellan gum (GG) has gained popularity as a versatile biomaterial for tissue engineering purposes. Here, we examine the modification strategies suitable for GG to overcome processing-related limitations. We emphasize the thorough assessment of the viscoelastic and mechanical properties of both precursor solutions and final hydrogels. The investigated modification strategies include purification, oxidation, reductive chain scission, and blending. We correlate polymer flow and hydrogel forming capabilities to viscosity-dependent methods including casting, injection and printing. Native GG and purified NaGG are shear thinning and feasible for printing, being similar in gelation and compression behavior. Oxidized GGox possesses reduced viscosity, higher toughness, and aldehydes as functional groups, while scissored GGsciss has markedly lower molecular weight. To exemplify extrudability, select modification products are printed using an extrusion-based bioprinter utilizing a crosslinker bath. Our robust modification strategies have widened the processing capabilities of GG without affecting its ability to form hydrogels.

Optical projection tomography as a quantitative tool for analysis of cell morphology and density in 3D hydrogels.

Assessing cell morphology and function, as well as biomaterial performance in cell cultures, is one of the key challenges in cell biology and tissue engineering (TE) research. In TE, there is an urgent need for methods to image actual three-dimensional (3D) cell cultures and access the living cells. This is difficult using established optical microscopy techniques such as wide-field or confocal microscopy. To address the problem, we have developed a new protocol using Optical Projection Tomography (OPT) to extract quantitative and qualitative measurements from hydrogel cell cultures. Using our tools, we demonstrated the method by analyzing cell response in three different hydrogel formulations in 3D with 1.5 mm diameter samples of: gellan gum (GG), gelatin functionalized gellan gum (gelatin-GG), and Geltrex. We investigated cell morphology, density, distribution, and viability in 3D living cells. Our results showed the usability of the method to quantify the cellular responses to biomaterial environment. We observed that an elongated morphology of cells, thus good material response, in gelatin-GG and Geltrex hydrogels compared with basic GG. Our results show that OPT has a sensitivity to assess in real 3D cultures the differences of cellular responses to the properties of biomaterials supporting the cells.

Impact of Glass Composition on Hydrolytic Degradation of Polylactide/Bioactive Glass Composites.

Understanding the degradation of a composite material is crucial for tailoring its properties based on the foreseen application. In this study, poly-L,DL-lactide 70/30 (PLA70) was compounded with silicate or phosphate bioactive glass (Si-BaG and P-BaG, respectively). The composite processing was carried out without excessive thermal degradation of the polymer and resulted in porous composites with lower mechanical properties than PLA70. The loss in mechanical properties was associated with glass content rather than the glass composition. The degradation of the composites was studied for 40 weeks in Tris buffer solution Adding Si-BaG to PLA70 accelerated the polymer degradation in vitro more than adding P-BaG, despite the higher reactivity of the P-BaG. All the composites exhibited a decrease in mechanical properties and increased hydrophilicity during hydrolysis compared to the PLA70. Both glasses dissolved through the polymer matrix with a linear, predictable release rate of ions. Most of the P-BaG had dissolved before 20 weeks in vitro, while there was still Si-BaG left after 40 weeks. This study introduces new polymer/bioactive glass composites with tailorable mechanical properties and ion release for bone regeneration and fixation applications.

Photocross-linkable Methacrylated Polypeptides and Polysaccharides for Casting, Injecting, and 3D Fabrication.

For modern tissue engineering, we need not only develop new hydrogels but also suitable processing methods for them. Polypeptides and polysaccharides are potential candidates because they can be methacrylated, processed before photocross-linking, and yielded into hydrogels with given shape and form. In this study, we successfully methacrylated collagen, gelatin, hyaluronan, and alginate to 30 and 60% degree of modification. We studied methacrylated compositions (i.e., precursors) to investigate their processability. The precursors of collagen and gelatin with 60% methacrylation exhibited suitable yield stress, shear-thinning properties, and fiber-forming capability for injecting and 3D bioprinting. On the contrary, the 30% methacrylated precursors had properties suitable for casting purposes. Our study also showed that the mechanical properties of hydrogels corresponded to the used photocross-linking conditions and the degree of modification. These results underline the importance of tunability of the precursors and resulting hydrogels according to the specific fabrication method and tissue engineering application.

A tube-source X-ray microtomography approach for quantitative 3D microscopy of optically challenging cell-cultured samples.

Development and study of cell-cultured constructs, such as tissue-engineering scaffolds or organ-on-a-chip platforms require a comprehensive, representative view on the cells inside the used materials. However, common characteristics of biomedical materials, for example, in porous, fibrous, rough-surfaced, and composite materials, can severely disturb low-energy imaging. In order to image and quantify cell structures in optically challenging samples, we combined labeling, 3D X-ray imaging, and in silico processing into a methodological pipeline. Cell-structure images were acquired by a tube-source X-ray microtomography device and compared to optical references for assessing the visual and quantitative accuracy. The spatial coverage of the X-ray imaging was demonstrated by investigating stem-cell nuclei inside clinically relevant-sized tissue-engineering scaffolds (5x13 mm) that were difficult to examine with the optical methods. Our results highlight the potential of the readily available X-ray microtomography devices that can be used to thoroughly study relative large cell-cultured samples with microscopic 3D accuracy.

Materials and Orthopedic Applications for Bioresorbable Inductively Coupled Resonance Sensors.

Bioresorbable passive resonance sensors based on inductor-capacitor (LC) circuits provide an auspicious sensing technology for temporary battery-free implant applications due to their simplicity, wireless readout, and the ability to be eventually metabolized by the body. In this study, the fabrication and performance of various LC circuit-based sensors are investigated to provide a comprehensive view on different material options and fabrication methods. The study is divided into sections that address different sensor constituents, including bioresorbable polymer and bioactive glass substrates, dissolvable metallic conductors, and atomic layer deposited (ALD) water barrier films on polymeric substrates. The manufactured devices included a polymer-based pressure sensor that remained pressure responsive for 10 days in aqueous conditions, the first wirelessly readable bioactive glass-based resonance sensor for monitoring the complex permittivity of its surroundings, and a solenoidal coil-based compression sensor built onto a polymeric bone fixation screw. The findings together with the envisioned orthopedic applications provide a reference point for future studies related to bioresorbable passive resonance sensors.

Evaluation of scaffold microstructure and comparison of cell seeding methods using micro-computed tomography-based tools.

Micro-computed tomography (micro-CT) provides a means to analyse and model three-dimensional (3D) tissue engineering scaffolds. This study proposes a set of micro-CT-based tools firstly for evaluating the microstructure of scaffolds and secondly for comparing different cell seeding methods. The pore size, porosity and pore interconnectivity of supercritical CO2 processed poly(l-lactide-co-ɛ-caprolactone) (PLCL) and PLCL/ß-tricalcium phosphate scaffolds were analysed using computational micro-CT models. The models were supplemented with an experimental method, where iron-labelled microspheres were seeded into the scaffolds and micro-CT imaged to assess their infiltration into the scaffolds. After examining the scaffold architecture, human adipose-derived stem cells (hASCs) were seeded into the scaffolds using five different cell seeding methods. Cell viability, number and 3D distribution were evaluated. The distribution of the cells was analysed using micro-CT by labelling the hASCs with ultrasmall paramagnetic iron oxide nanoparticles. Among the tested seeding methods, a forced fluid flow-based technique resulted in an enhanced cell infiltration throughout the scaffolds compared with static seeding. The current study provides an excellent set of tools for the development of scaffolds and for the design of 3D cell culture experiments.

Bioactive glass ions for in vitro osteogenesis and microvascularization in gellan gum-collagen hydrogels.

Lack of bone grafts appeals for bone augmentation solutions. We aimed at osteogenic differentiation of human adipose stem cells (hASCs) and microvascularization in coculture with human umbilical vein endothelial cells (HUVECs) embedded in three-dimensional (3D) gellan gum (GG) and collagen type I (COL) hydrogel mixture. We compared endothelial growth medium-2 (EGM-2) and bioactive glass extract-based endothelial and osteogenic medium (BaG EM-OM) for vascularized bone-like graft development in vitro. Cell viability, cell number, and osteogenic and endothelial gene expression were analyzed. Mineralized hydroxyapatite residues, immunocytochemical staining of endothelial marker CD31 production and late osteogenic marker osteocalcin were imaged. With both media, good cell viability was observed within 3D hydrogel. EGM-2 condition induced significantly higher cell number compared to BaG EM-OM condition at both 7 and 14 days. Interestingly, both media supported osteogenic as well as endothelial marker gene expression. Moreover, formation of reticulated cellular structures was observed in both EGM-2 and BaG EM-OM conditions. However, hydroxyapatite mineralization and strong osteocalcin staining were detected only in BaG EM-OM condition. Importantly, strong production of CD31 and elongated tube-like structures were apparent in EGM-2 culture alone. In conclusion, we demonstrated efficient hASC osteogenic differentiation and microvessel-like network formation in coculture with HUVECs.

Co-culture of human induced pluripotent stem cell-derived retinal pigment epithelial cells and endothelial cells on double collagen-coated honeycomb films.

In vitro cell culture models representing the physiological and pathological features of the outer retina are urgently needed. Artificial tissue replacements for patients suffering from degenerative retinal diseases are similarly in great demand. Here, we developed a co-culture system based solely on the use of human induced pluripotent stem cell (hiPSC)-derived cells. For the first time, hiPSC-derived retinal pigment epithelium (RPE) and endothelial cells (EC) were cultured on opposite sides of porous polylactide substrates prepared by breath figures (BF), where both surfaces had been collagen-coated by Langmuir-Schaefer (LS) technology. Small modifications of casting conditions during material preparation allowed the production of free-standing materials with distinct porosity, wettability and ion diffusion capacity. Complete pore coverage was achieved by the collagen coating procedure, resulting in a detectable nanoscale topography. Primary retinal endothelial cells (ACBRI181) and umbilical cord vein endothelial cells (hUVEC) were utilised as EC references. Mono-cultures of all ECs were prepared for comparison. All tested materials supported cell attachment and growth. In mono-culture, properties of the materials had a major effect on the growth of all ECs. In co-culture, the presence of hiPSC-RPE affected the primary ECs more significantly than hiPSC-EC. In consistency, hiPSC-RPE were also less affected by hiPSC-EC than by the primary ECs. Finally, our results show that the modulation of the porosity of the materials can promote or prevent EC migration. In short, we showed that the behaviour of the cells is highly dependent on the three main variables of the study: the presence of a second cell type in co-culture, the source of endothelial cells and the biomaterial properties. The combination of BF and LS methodologies is a powerful strategy to develop thin but stable materials enabling cell growth and modulation of cell-cell contact. STATEMENT OF SIGNIFICANCE: Artificial blood-retinal barriers (BRB), mimicking the interface at the back of the eye, are urgently needed as physiological and disease models, and for tissue transplantation targeting patients suffering from degenerative retinal diseases. Here, we developed a new co-culture model based on thin, biodegradable porous films, coated on both sides with collagen, one of the main components of the natural BRB, and cultivated endothelial and retinal pigment epithelial cells on opposite sides of the films, forming a three-layer structure. Importantly, our hiPSC-EC and hiPSC-RPE co-culture model is the first to exclusively use human induced pluripotent stem cells as cell source, which have been widely regarded as an practical candidate for therapeutic applications in regenerative medicine.

Effect of Melt-Derived Bioactive Glass Particles on the Properties of Chitosan Scaffolds.

This study reports on the processing of three-dimensional (3D) chitosan/bioactive glass composite scaffolds. On the one hand, chitosan, as a natural polymer, has suitable properties for tissue engineering applications but lacks bioactivity. On the other hand, bioactive glasses are known to be bioactive and to promote a higher level of bone formation than any other biomaterial type. However, bioactive glasses are hard, brittle, and cannot be shaped easily. Therefore, in the past years, researchers have focused on the processing of new composites. Difficulties in reaching composite materials made of polymer (synthetic or natural) and bioactive glass include: (i) The high glass density, often resulting in glass segregation, and (ii) the fast bioactive glass reaction when exposed to moisture, leading to changes in the glass reactivity and/or change in the polymeric matrix. Samples were prepared with 5, 15, and 30 wt% of bioactive glass S53P4 (BonAlive ®), as confirmed using thermogravimetric analysis. MicrO-Computed tomography and optical microscopy revealed a flaky structure with porosity over 80%. The pore size decreased when increasing the glass content up to 15 wt%, but increased back when the glass content was 30 wt%. Similarly, the mechanical properties (in compression) of the scaffolds increased for glass content up to 15%, but decreased at higher loading. Ions released from the scaffolds were found to lead to precipitation of a calcium phosphate reactive layer at the scaffold surface. This is a first indication of the potential bioactivity of these materials. Overall, chitosan/bioactive glass composite scaffolds were successfully produced with pore size, machinability, and ability to promote a calcium phosphate layer, showing promise for bone tissue engineering and the mechanical properties can justify their use in non-load bearing applications.

Design of modular gellan gum hydrogel functionalized with avidin and biotinylated adhesive ligands for cell culture applications.

This article proposes the coupling of the recombinant protein avidin to the polysaccharide gellan gum to create a modular hydrogel substrate for 3D cell culture and tissue engineering. Avidin is capable of binding biotin, and thus biotinylated compounds can be tethered to the polymer network to improve cell response. The avidin is successfully conjugated to gellan gum and remains functional as shown with fluorescence titration and electrophoresis (SDS-PAGE). Self-standing hydrogels were formed using bioamines and calcium chloride, yielding long-term stability and adequate stiffness for 3D cell culture, as confirmed with compression testing. Human fibroblasts were successfully cultured within the hydrogel treated with biotinylated RGD or biotinylated fibronectin. Moreover, human bone marrow stromal cells were cultured with hydrogel treated with biotinylated RGD over 3 weeks. We demonstrate a modular and inexpensive hydrogel scaffold for cell encapsulation that can be equipped with any desired biotinylated cell ligand to accommodate a wide range of cell types.

Polyethylene Terephthalate Textiles Enhance the Structural Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have the potential to serve as a model for human cardiomyocytes. However, hiPSC-CMs are still considered immature. CMs differentiated from hiPSCs more resemble fetal than adult cardiomyocytes. Putative factors enhancing maturation include in vitro culture duration, culture surface topography, and mechanical, chemical, and electrical stimulation. Stem cell-derived cardiomyocytes are traditionally cultured on glass surfaces coated with extracellular matrix derivatives such as gelatin. hiPSC-CMs are flat and round and their sarcomeres are randomly distributed and unorganized. Morphology can be enhanced by culturing cells on surfaces providing topographical cues to the cells. In this study, a textile based-culturing method used to enhance the maturation status of hiPSC-CMs is presented. Gelatin-coated polyethylene terephthalate (PET)-based textiles were used as the culturing surface for hiPSC-CMs and the effects of the textiles on the maturation status of the hiPSC-CMs were assessed. The hiPSC-CMs were characterized by analyzing their morphology, sarcomere organization, expression of cardiac specific genes, and calcium handling. We show that the topographical cues improve the structure of the hiPSC-CMs in vitro. Human iPSC-CMs grown on PET textiles demonstrated improved structural properties such as rod-shape structure and increased sarcomere orientation.

Screening of Hydrogels for Human Pluripotent Stem Cell-Derived Neural Cells: Hyaluronan-Polyvinyl Alcohol-Collagen-Based Interpenetrating Polymer Network Provides an Improved Hydrogel Scaffold.

There is a clear need for novel in vitro models, especially for neuronal applications. Development of in vitro models is a multiparameter task consisting of cell-, biomaterial-, and environment-related parameters. Here, three different human origin neuronal cell sources are studied and cultured in various hydrogel 3D scaffolds. For the efficient evaluation of complex results, an indexing method for data is developed and used in principal component analysis (PCA). It is found that no single hydrogel is superior to other hydrogels, and collagen I (Col1) and hyaluronan-poly(vinyl alcohol) (HA1-PVA) gels are combined into an interpenetrating network (IPN) hydrogel. The IPN gel combines cell supportiveness of the collagen gel and stability of the HA1-PVA gel. Moreover, cell adhesion is studied in particular and it is found that adhesion of neurons differs from that observed for fibroblasts. In conclusion, the HA1-PVA-col1 hydrogel is a suitable scaffold for neuronal cells and supports adhesion formation in 3D.

Mechanically Biomimetic Gelatin-Gellan Gum Hydrogels for 3D Culture of Beating Human Cardiomyocytes.

To promote the transition of cell cultures from 2D to 3D, hydrogels are needed to biomimic the extracellular matrix (ECM). One potential material for this purpose is gellan gum (GG), a biocompatible and mechanically tunable hydrogel. However, GG alone does not provide attachment sites for cells to thrive in 3D. One option for biofunctionalization is the introduction of gelatin, a derivative of the abundant ECM protein collagen. Unfortunately, gelatin lacks cross-linking moieties, making the production of self-standing hydrogels difficult under physiological conditions. Here, we explore the functionalization of GG with gelatin at biologically relevant concentrations using semiorthogonal, cytocompatible, and facile chemistry based on hydrazone reaction. These hydrogels exhibit mechanical behavior, especially elasticity, which resembles the cardiac tissue. The use of optical projection tomography for 3D cell microscopy demonstrates good cytocompatibility and elongation of human fibroblasts (WI-38). In addition, human-induced pluripotent stem cell-derived cardiomyocytes attach to the hydrogels and recover their spontaneous beating in 24 h culture. Beating is studied using in-house-built phase contrast video analysis software, and it is comparable with the beating of control cardiomyocytes under regular culture conditions. These hydrogels provide a promising platform to transition cardiac tissue engineering and disease modeling from 2D to 3D.