Mechanically primed cells transfer memory to fibrous matrices for invasion across environments of distinct stiffness and dimensionality.

Cells sense and migrate across mechanically dissimilar environments throughout development and disease progression. However, it remains unclear whether mechanical memory of past environments empowers cells to navigate new, three-dimensional extracellular matrices. Here, we show that cells previously primed on stiff, compared with soft, matrices generate a higher level of forces to remodel collagen fibers and promote invasion. This priming advantage persists in dense or stiffened collagen. We explain this memory-dependent, cross-environment cell invasion through a lattice-based model wherein stiff-primed cellular forces remodel collagen and minimize energy required for future cell invasion. According to our model, cells transfer their mechanical memory to the matrix via collagen alignment and tension, and this remodeled matrix informs future cell invasion. Thus, memory-laden cells overcome mechanosensing of softer or challenging future environments via a cell-matrix transfer of memory. Consistent with model predictions, depletion of yes-associated protein destabilizes the cellular memory required for collagen remodeling before invasion. We release tension in collagen fibers via laser ablation and disable fiber remodeling by lysyl-oxidase inhibition, both of which disrupt cell-to-matrix transfer of memory and hamper cross-environment invasion. These results have implications for cancer, fibrosis, and aging, where a potential cell-to-matrix transfer of mechanical memory of cells may generate a prolonged cellular response.

A novel 3D vascular assay for evaluating angiogenesis across porous membranes.

Microfluidic technology has been extensively applied to model the functional units of human organs and tissues. Since vasculature is a key component of any functional tissue, a variety of techniques to mimic vasculature in vitro have been developed to address complex physiological and pathological processes in 3D tissues. Herein, we developed a novel, in vitro, microfluidic-based model to probe microvasculature growth into and across implanted porous membranes. Using ePTFE and polycarbonate as examples, we characterize the vascularization potential of these thin porous membranes using this device. This tool will allow for the assessment of porous materials early in their development, prior to their use for encapsulating implants or drugs, while minimizing the need for animal studies. Employing quantitative morphometric analysis and measurements of vascular permeability, we demonstrate our model to be an effective platform for evaluation of angiogenic potential of an implanted membrane biomaterial. Results show that endothelial cells can either migrate as single cells or form continuous sprouts across porous membranes, which is a material structure-dependent behavior. Our model is advantageous over conventional Transwell assays as it is amenable to quantitative assessment of vascular sprouting in 3D, and in contrast to animal models it can be employed more efficiently and with real-time assessment capabilities. This new tool could be applied either to test the suitability of a wide range of biomaterials for implantation or to screen different pro-angiogenic factors for therapeutic applications, and will advance the design of new biomaterials.

Longer collagen fibers trigger multicellular streaming on soft substrates via enhanced forces and cell-cell cooperation.

Grouped cells often leave large cell colonies in the form of narrow multicellular streams. However, it remains unknown how collective cell streaming exploits specific matrix properties, like stiffness and fiber length. It is also unclear how cellular forces, cell-cell adhesion and velocities are coordinated within streams. To independently tune stiffness and collagen fiber length, we developed new hydrogels and discovered invasion-like streaming of normal epithelial cells on soft substrates coated with long collagen fibers. Here, streams arise owing to a surge in cell velocities, forces, YAP activity and expression of mesenchymal marker proteins in regions of high-stress anisotropy. Coordinated velocities and symmetric distribution of tensile and compressive stresses support persistent stream growth. Stiff matrices diminish cell-cell adhesions, disrupt front-rear velocity coordination and do not promote sustained fiber-dependent streaming. Rac inhibition reduces cell elongation and cell-cell cooperation, resulting in a complete loss of streaming in all matrix conditions. Our results reveal a stiffness-modulated effect of collagen fiber length on collective cell streaming and unveil a biophysical mechanism of streaming governed by a delicate balance of enhanced forces, monolayer cohesion and cell-cell cooperation.This article has an associated First Person interview with the first authors of the paper.

Predicting Collective Migration of Cell Populations Defined by Varying Repolarization Dynamics.

Collective migration of heterogeneous cell populations is an essential aspect of fundamental biological processes, including morphogenesis, wound healing, and tumor invasion. Through experiments and modeling, it has been shown that cells attain front-rear polarity, generate forces, and form adhesions to migrate. However, it remains unclear how the ability of individual cells in a population to dynamically repolarize themselves into new directions could regulate the collective response. We present a vertex-based model in which each deformable cell randomly chooses a new polarization direction after every defined time interval, elongates, proportionally generates forces, and causes collective migration. Our simulations predict that cell types that repolarize at longer time intervals attain more elongated shapes, migrate faster, deform the cell sheet, and roughen the leading edge. By imaging collectively migrating epithelial cell monolayers at high temporal resolution, we found longer repolarization intervals and elongated shapes of cells at the leading edge compared to those within the monolayer. Based on these experimental measurements and simulations, we defined aggressive mutant leader cells by long repolarization interval and minimal intercellular contact. The cells with frequent and random repolarization were defined as normal cells. In simulations with uniformly dispersed leader cells in a normal cell population at a 1:10 ratio, the resulting migration and deformation of the heterogeneous cell sheet remained low. However, when the 10% mutant leaders were placed only at the leading edge, we predicted a rise in the migration of an otherwise normal cell sheet. Our model predicts that a repolarization-based definition of leader cells and their placement within a healthy population can generate myriad modes of collective cell migration, which can enhance our understanding of collective cell migration in disease and development.

Encapsulation of Rat Bone Marrow Derived Mesenchymal Stem Cells in Alginate Dialdehyde/Gelatin Microbeads with and without Nanoscaled Bioactive Glass for In Vivo Bone Tissue Engineering.

Alginate dialdehyde (ADA), gelatin, and nano-scaled bioactive glass (nBG) particles are being currently investigated for their potential use as three-dimensional scaffolding materials for bone tissue engineering. ADA and gelatin provide a three-dimensional scaffold with properties supporting cell adhesion and proliferation. Combined with nanocristalline BG, this composition closely mimics the mineral phase of bone. In the present study, rat bone marrow derived mesenchymal stem cells (MSCs), commonly used as an osteogenic cell source, were evaluated after encapsulation into ADA-gelatin hydrogel with and without nBG. High cell survival was found in vitro for up to 28 days with or without addition of nBG assessed by calcein staining, proving the cell-friendly encapsulation process. After subcutaneous implantation into rats, survival was assessed by DAPI/TUNEL fluorescence staining. Hematoxylin-eosin staining and immunohistochemical staining for the macrophage marker ED1 (CD68) and the endothelial cell marker lectin were used to evaluate immune reaction and vascularization. After in vivo implantation, high cell survival was found after 1 week, with a notable decrease after 4 weeks. Immune reaction was very mild, proving the biocompatibility of the material. Angiogenesis in implanted constructs was significantly improved by cell encapsulation, compared to cell-free beads, as the implanted MSCs were able to attract endothelial cells. Constructs with nBG showed higher numbers of vital MSCs and lectin positive endothelial cells, thus showing a higher degree of angiogenesis, although this difference was not significant. These results support the use of ADA/gelatin/nBG as a scaffold and of MSCs as a source of osteogenic cells for bone tissue engineering. Future studies should however improve long term cell survival and focus on differentiation potential of encapsulated cells in vivo.

Hydrogel matrices based on elastin and alginate for tissue engineering applications.

Hydrogels from natural polymers are widely used in tissue engineering due to their unique properties, especially when regarding the cell environment and their morphological similarity to the extracellular matrix (ECM) of native tissues. In this study, we describe the production and characterization of novel hybrid hydrogels composed of alginate blended with elastin from bovine neck ligament. The properties of elastin as a component of the native ECM were combined with the excellent chemical and mechanical stability as well as biocompatibility of alginate to produce two hybrid hydrogels geometries, namely 2D films obtained using sonication treatment and 3D microcapsules produced by pressure-driven extrusion. The resulting blend hydrogels were submitted to an extensive physico-chemical characterization. Furthermore, the biological compatibility of these materials was assessed using normal human dermal fibroblasts, indicating the suitability of this blend for soft tissue engineering.

Direct Micropatterning of Extracellular Matrix Proteins on Functionalized Polyacrylamide Hydrogels Shows Geometric Regulation of Cell-Cell Junctions.

Microcontact printing of extracellular matrix (ECM) proteins in defined regions of a substrate allows spatial control over cell attachment and enables the study of cellular response to irregular ECM geometries. Over the past decade, numerous micropatterning techniques have emerged that conjugate ECM proteins on hydrogel substrates of tunable stiffness, which have revealed a range of cellular responses to varying matrix stiffness and geometry. However, micropatterning of ECM proteins on polyacrylamide (PA) hydrogel remains inconsistent due to its unreliable conjugation with the commonly used protein cross-linkers, particularly at low stiffness. To address these problems, we developed a micropatterning technique in which the PA gel is functionalized by incorporating oxidized N-hydroxyethylacrylamide, which allows direct protein binding through reactive aldehyde groups without any exogenous cross-linkers. As a result, a uniform and consistent protein transfer onto the hydrogel substrates of defined geometries is achieved, even for soft PA gels. We formed square, rectangular, and triangular patterns of two constant areas on soft and stiff PA gels that provide large and small adhesive areas for the MCF10A human mammary epithelial cell pairs. We measured intercellular E-cadherin (E-cad) expression and found that cell-cell junctions could be deteriorated independently by either the stiff ECM of any shape or the elongated cell morphology, accompanied by increased cell-generated tractions, on rectangular soft ECM patterns. Inhibition of nonmuscle myosin II reduced the E-cad junctional localization in cell pairs. When the cell spreading was restricted by reducing the adhesive area of the patterns, we observed an overall rise in E-cad expression at cell-cell junctions. Our findings present an improved micropatterning technique which reveals a geometric regulation of cell-cell junctions in epithelial cell pairs.

Oxidized Alginate-Gelatin Hydrogel: A Favorable Matrix for Growth and Osteogenic Differentiation of Adipose-Derived Stem Cells in 3D.

Alginate-based hydrogels are extensively used matrices for cell encapsulation, but they need to be modified to recapitulate chemical, microstructural, and mechanical properties of the native extracellular matrix. Like other cell types, mesenchymal stem cells exhibit rounded and clustered morphologies when they are embedded in alginate hydrogels. In this study, we use covalently cross-linked oxidized alginate-gelatin hydrogels to encapsulate human adipose-derived stem cells in order to investigate cell growth, viability, and morphology during osteogenic differentiation taking advantage of the different physicochemical properties of this modified alginate-based hydrogel in comparison to those of the pristine alginate hydrogel. We investigate the effect of hydrogel compositions on stem cell behavior in 3D. Higher viability and the spreading morphology of encapsulated cells with interconnected networks were observed in high gelatin containing compositions. More filopodial protrusions from multicellular nodules were noticed during osteogenic differentiation in the hydrogels having a high amount of gelatin, confirming their suitability for cell encapsulation and bone tissue engineering applications.

Bioplotting of a bioactive alginate dialdehyde-gelatin composite hydrogel containing bioactive glass nanoparticles.

Alginate dialdehyde-gelatin (ADA-GEL) constructs incorporating bioactive glass nanoparticles (BGNPs) were produced by biofabrication to obtain a grid-like highly-hydrated composite. The material could induce the deposition of an apatite layer upon immersion in a biological-like environment to sustain cell attachment and proliferation. Composites were formulated with different concentrations of BGNPs synthetized from a sol-gel route, namely 0.1% and 0.5% (w/v). Strontium doped BGNPs were also used. EDS analysis suggested that the BGNPs loading promoted the growth of bone-like apatite layer on the surface when the constructs were immersed in a simulated body fluid. Moreover, the composite constructs could incorporate with high efficiency ibuprofen as a drug model. Furthermore, the biofabrication process allowed the successful incorporation of MG-63 cells into the composite material. Cells were distributed homogeneously within the hydrogel composite, and no differences were found in cell viability between ADA-GEL and the composite constructs, proving that the addition of BGNPs did not influence cell fate. Overall, the composite material showed potential for future applications in bone tissue engineering.

Biofabrication of 3D Alginate-Based Hydrogel for Cancer Research: Comparison of Cell Spreading, Viability, and Adhesion Characteristics of Colorectal HCT116 Tumor Cells.

Hydrogels are an important class of biomaterials as they could mimic the extracellular matrix (ECM). Among the naturally occurring biopolymers, alginate and gelatin are extensively used for many biomedical applications. For developing biofabrication constructs as three-dimensional (3D) cell culture models, realistic imaging of cell spreading and proliferation inside the hydrogels represents a major challenge. Therefore, we aimed to establish a system that can mimic the structural architecture, composition, and biological functions of the ECM for cancer research approaches. For this, we compared the cell behavior of human colon cancer HCT116 cells in two biofabricated hydrogels as follows: pure alginate and cross-linked alginate-gelatin (ADA-GEL) matrixes. Our data indicate that cells from the ADA-GEL matrix showed highest proliferation and cellular networks through the material. Analyzing the mRNA expression of several integrins of cells cultured inside of the matrix, we showed that mRNA expression of integrin subunits differed based on the cell focal adhesion characteristics. Furthermore, we showed that recultured ADA-GEL immobilized cells do not differ from parental HCT116 cells regarding migration and proliferation capabilities. Comparing adhesion and other phenotypic characteristics of HCT116 tumor cells, we suggest that ADA-GEL hydrogel is a more suitable 3D system than pure alginate and seems to optimally mimic the physiological behavior of the tumor microenvironment. For the first time, we present a functional 3D hydrogel construct for colon cancer cells, which are supporting their physiological cell attachment, spreading, and viability. We strongly believe that it will be applicable as a suitable in vitro 3D tumor model to study different aspects of tumor cell behavior.

Soft-matrices based on silk fibroin and alginate for tissue engineering.

Soft tissue regeneration requires the use of matrices that exhibit adequate mechanical properties as well as the ability to supply nutrients and oxygen, and to remove metabolic bio-products. In this work, we describe the development of hydrogels based on the blend between alginate (Alg) and silk fibroin (SF). Herein, we report two main strategies to combine cells with biomaterials: cells are either seeded onto prefabricated hydrogels films (2D), or encapsulated during hydrogel microcapsules formation (3D). Both geometries were successfully produced and characterized. FTIR results indicated a change of conformation of SF from random coil to ß-sheet after hydrogel formation. The thermal degradation behavior of films and microcapsules fabricated from Alg, and Alg/SF was dependent on the hydrogel composition and on the geometry of the samples. The presence of SF caused decreased water uptake ability and affected the degradation behavior. Mechanical tests showed that addition of SF promotes an increase in storage modulus, leading to a stiffer material as compared with pure Alg (6 times higher stiffness). Moreover, the in vitro cell-material interaction on Alg/SF hydrogels of different geometries was investigated using human umbilical vein endothelial cells (HUVECs). Viability, attachment, spreading and proliferation of HUVECs were significantly increased on Alg/SF hydrogels compared to neat Alg. These findings indicate that Alg/SF hydrogel is a promising material for the biomedical applications in tissue-engineering and regeneration.

Evaluation of hydrogel matrices for vessel bioplotting: Vascular cell growth and viability.

Developing matrices biocompatible with vascular cells is one of the most challenging tasks in tissue engineering. Here, we compared the growth of vascular cells on different hydrogels as potential materials for bioplotting of vascular tissue. Formulations containing alginate solution (Alg, 2%, w/v) blended with protein solutions (silk fibroin, gelatin, keratin, or elastin) at 1% w/v were prepared. Human umbilical vein endothelial cells (ECs), smooth muscle cells (SMCs), and fibroblasts were cultivated on hydrogels for 7 days. Cell number and morphology was visualised using fluorescent staining at day 3 and 7. Cell metabolic activity was analysed using WST assay. Compared to pure Alg, Alg/keratin, Alg/gelatin and Alg/silk fibroin provided superb surfaces for ECs, supporting their attachment, growth, spreading and metabolic activity. SMCs showed best colonization and growth on Alg/silk fibroin and Alg/keratin hydrogels, whereas on elastin-containing hydrogels, cell clustering was observed. Fibroblasts growth was enhanced on Alg/elastin, and strongly improved on silk fibroin- and keratin-containing hydrogels. In contrast to the previous studies with alginate dialdehyde-gelatin crosslinked gels, Alg/gelatin blend hydrogels provided a less favourable scaffold for fibroblasts. Taken together, the most promising results were obtained with silk fibroin- and keratin-containing hydrogels, which supported the growth of all types of vascular cells. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 577-585, 2016.

Designing Porous Bone Tissue Engineering Scaffolds with Enhanced Mechanical Properties from Composite Hydrogels Composed of Modified Alginate, Gelatin, and Bioactive Glass.

The combination of biodegradable polymers and bioactive inorganic materials is being widely used for designing bone tissue engineering scaffolds. Here we report a composite hydrogel system composed of bioactive glass incorporated in covalently cross-linked oxidized alginate-gelatin hydrogel (ADA-GEL) for designing porous scaffolds with tunable stiffness and degradability using freeze-drying technique. Because of the presence of bioactive glass, the cross-linking kinetic and cross-linking degree of the hydrogels are significantly increased, which is the main factor for the measured enhanced mechanical strength of the bioactive glass containing ADA-GEL scaffolds. The hydrogels with high cross-linking degree exhibit low protein release profile and low degradability. Apatite formation on bioactive glass containing hydrogel-based scaffolds is confirmed by FTIR. Bone marrow-derived stromal cell growth is promoted in pristine ADA-GEL and 1% bioactive glass containing ADA-GEL scaffolds compared to the scaffolds of pure alginate, alginate-gelatin blended hydrogel, and 5% bioactive glass containing ADA-GEL. Initial studies indicated that the scaffolds, especially without bioactive glass, support osteogenic differentiation of murine bone marrow stromal cell line in the absence of foreign osteogenic stimulating supplements; however, they exhibit low levels of osteogenic expression.

Alginate-based hydrogels with improved adhesive properties for cell encapsulation.

Hydrogel-based biomaterials are ideal scaffolding matrices for microencapsulation, but they need to be modified to resemble the mechanical, structural and chemical properties of the native extracellular matrix. Here, we compare the mechanical properties and the degradation behavior of unmodified and modified alginate hydrogels in which cell adhesive functionality is conferred either by blending or covalently cross-linking with gelatin. Furthermore, we measure the spreading and proliferation of encapsulated osteoblast-like MG-63 cells. Alginate hydrogels covalently crosslinked with gelatin show the highest degree of cell adhesion, spreading, migration, and proliferation, as well as a faster degradation rate, and are therefore a particularly suitable material for microencapsulation.

Evaluation of an alginate-gelatine crosslinked hydrogel for bioplotting.

Using additive manufacturing to create hydrogel scaffolds which incorporate homogeneously distributed, immobilized cells in the context of biofabrication approaches represents an emerging and expanding field in tissue engineering. Applying hydrogels for additive manufacturing must consider the material processing properties as well as their influence on the immobilized cells. In this work alginate-dialdehyde (ADA), a partially oxidized alginate, was used as a basic material to improve the physico-chemical properties of the hydrogel for cell immobilization. At first, the processing ability of the gel using a bioplotter and the compatibility of the process with MG-63 osteoblast like cells were investigated. The metabolic and mitochondrial activities increased at the beginning of the incubation period and they balanced at a relatively high level after 14-28 days of incubation. During this incubation period the release of vascular endothelial growth factor-A also increased. After 28 days of incubation the cell morphology showed a spreading morphology and cells were seen to move out of the scaffold struts covering the whole scaffold structure. The reproducible processing capability of alginate-gelatine (ADA-GEL) and the compatibility with MG-63 cells were proven, thus the ADA-GEL material is highlighted as a promising matrix for applications in biofabrication.

Combining collagen and bioactive glasses for bone tissue engineering: a review.

Collagen (COL), the most abundant protein in mammals, offers a wide range of attractive properties for biomedical applications which are the result of its biocompatibility and high affinity to water. However, due to the relative low mechanical properties of COL its applications are still limited. To tackle this disadvantage of COL, especially in the field of bone tissue engineering, COL can be combined with bioactive inorganic materials in a variety of composite systems. One of such systems is the collagen-bioactive glass (COL-BG) composite family, which is the theme of this Review. BG fillers can increase compressive strength and stiffness of COL-based structures. This article reviews the relevant literature published in the last 15 years discussing the fabrication of a variety of COL-BG composites. In vitro cell studies have demonstrated the osteogenic, odontogenic, and angiogenic potential of these composite systems, which has been confirmed by stimulating specific biochemical indicators of relevant cells. Bony integration and connective tissue vessel formation have also been studied by implantation of the composites in vivo. Areas of future research in the field of COL-BG systems, based on current challenges, and gaps in knowledge are highlighted.

Evaluation of fibroblasts adhesion and proliferation on alginate-gelatin crosslinked hydrogel.

Due to the relatively poor cell-material interaction of alginate hydrogel, alginate-gelatin crosslinked (ADA-GEL) hydrogel was synthesized through covalent crosslinking of alginate di-aldehyde (ADA) with gelatin that supported cell attachment, spreading and proliferation. This study highlights the evaluation of the physico-chemical properties of synthesized ADA-GEL hydrogels of different compositions compared to alginate in the form of films. Moreover, in vitro cell-material interaction on ADA-GEL hydrogels of different compositions compared to alginate was investigated by using normal human dermal fibroblasts. Viability, attachment, spreading and proliferation of fibroblasts were significantly increased on ADA-GEL hydrogels compared to alginate. Moreover, in vitro cytocompatibility of ADA-GEL hydrogels was found to be increased with increasing gelatin content. These findings indicate that ADA-GEL hydrogel is a promising material for the biomedical applications in tissue-engineering and regeneration.

Behavior of encapsulated MG-63 cells in RGD and gelatine-modified alginate hydrogels.

Achieving cell spreading and proliferation inside hydrogels that are compatible with microencapsulation technology represents a major challenge for tissue engineering scaffolding and for the development of three-dimensional cell culture models. In this study, microcapsules of 650-900 µm in diameter were fabricated from oxidized alginate covalently cross-linked with gelatine (AlGel). Schiff's base bond formed in AlGel, detected by Fourier transform infrared spectroscopy, which confirmed the cross-linking of oxidized alginate with gelatine. Biological properties of alginate based hydrogels were studied by comparing the viability and morphology of MG-63 osteosarcoma cells encapsulated in gelatine and RGD-modified alginate. We hypothesized that the presence of gelatine and RGD will support cell adhesion and spreading inside the microcapsules and finally, also vascular endothelial growth factor (VEGF) secretion. After 4 days of incubation, cells formed extensive cortical protrusions and after 2 weeks they proliferated, migrated, and formed cellular networks through the AlGel material. In contrast, cells encapsulated in pure alginate and in RGD-modified alginate formed spherical aggregates with limited cell mobility and VEGF secretion. Metabolic activity was doubled after 5 days of incubation, making AlGel a promising material for cell encapsulation.

Fabrication of alginate-gelatin crosslinked hydrogel microcapsules and evaluation of the microstructure and physico-chemical properties.

Microencapsulation of cells by using biodegradable hydrogels offers numerous attractive features for a variety of biomedical applications including tissue engineering. This study highlights the fabrication of microcapsules from an alginate-gelatin crosslinked hydrogel (ADA-GEL) and presents the evaluation of the physico-chemical properties of the new microcapsules which are relevant for designing suitable microcapsules for tissue engineering. Alginate di-aldehyde (ADA) was synthesized by periodate oxidation of alginate which facilitates crosslinking with gelatin through Schiff's base formation between the free amino groups of gelatin and the available aldehyde groups of ADA. Formation of Schiff's base in ADA-GEL and aldehyde groups in ADA was confirmed by FTIR and NMR spectroscopy, respectively. Thermal degradation behavior of films and microcapsules fabricated from alginate, ADA and ADA-GEL was dependent on the hydrogel composition. The gelation time of ADA-GEL was found to decrease with increasing gelatin content. The swelling ratio of ADA-GEL microcapsules of all compositions was significantly decreased, whereas the degradability was found to increase with the increase of gelatin ratio. The surface morphology of the ADA-GEL microcapsules was totally different from that of alginate and ADA microcapsules, observed by SEM. Two different buffer solutions (with and without calcium salt) have an influence on the stability of microcapsules which had a significant effect on the gelatin release profile of ADA-GEL microcapsules in these two buffer solutions.

Hybrid hydrogels based on keratin and alginate for tissue engineering.

Novel hybrid hydrogels based on alginate and keratin were successfully produced for the first time. The self-assembly properties of keratin, and its ability to mimic the extracellular matrix were combined with the excellent chemical and mechanical stability and biocompatibility of alginate to produce 2D and 3D hybrid hydrogels. These hybrid hydrogels were prepared using two different approaches: sonication, to obtain 2D hydrogels, and a pressure-driven extrusion technique to produce 3D hydrogels. All results indicated that the composition of the hydrogels had a significant effect on their physical properties, and that they can easily be tuned to obtain materials suitable for biological applications. The cell-material interaction was assessed through the use of human umbilical vein endothelial cells, and the results demonstrated that the alginate/keratin hybrid biomaterials supported cell attachment, spreading and proliferation. The results proved that such novel hybrid hydrogels might find applications as scaffolds for soft tissue regeneration.