Tobias Zehnder

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 Schiffs base formation between the free amino groups of gelatin and the available aldehyde groups of ADA. Formation of Schiffs 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.

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.

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.

Additive manufacturing of cell-loaded alginate enriched with alkaline phosphatase for bone tissue engineering application

Abstract Hydrogels are gaining interest as scaffolds for bone tissue regeneration due to ease of incorporation of cells and biological molecules such as enzymes. Mineralization of hydrogels, desirable for bone tissue regeneration applications, may be achieved enzymatically by incorporation of alkaline phosphatase (ALP). Additive manufacturing techniques such as bioplotting enable the layer-by-layer creation of three-dimensional hydrogel scaffolds with highly defined geometry and internal architecture. In this study, we present a novel method to produce macroporous hydrogel scaffolds in combination with cell-loaded capsule-containing struts by 3D bioplotting. This approach enables loading of the capsules and strut phases with different cells and/or bioactive substances and hence makes compartmentalization within a scaffold possible. 3D porous alginate scaffolds enriched with ALP and MG-63 osteoblast-like cells were produced by bioplotting struts of alginate which were loaded with pre-fabricated alginate capsules. Two combinations were compared, namely ALP in the struts and cells in the capsules and vice-versa. Both combinations were cytocompatible for cells and mineralization of scaffolds could be detected in both cases, according to an OsteoImage staining. ALP had no adverse effect on cytocompatibility and enhanced mitochondrial activity.

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.

Macromolecular interactions in alginate–gelatin hydrogels regulate the behavior of human fibroblasts:

Due to the presence of tripeptide arginine–glycine–aspartic acid, gelatin is considered a very promising additive material to improve the cytocompatibility of alginate-based hydrogels. Two different strategies, physical blending and chemical crosslinking with gelatin, are used in this study to modify alginate hydrogel. As the intermolecular interactions between the polysaccharide and protein in the resulting physically blended and chemically crosslinked hydrogels are different, significant differences in the properties of these hydrogel types, regarding especially their surface topography, degradation kinetics, mechanical properties, and protein release behavior, are observed. Cellular behavior on both types of alginate–gelatin hydrogels is investigated using primary human dermal fibroblasts to elucidate the effects of the different structural, mechanical, and degradation properties of the produced hydrogels on fibroblast attachment and growth. The hydrogel that is chemically crosslinked with gelatin exhibits the highest degree of cytocompatibility regarding adhesion, proliferation, metabolic activity, and morphology of growing fibroblasts.

Evaluation of cell inkjet printing technique for biofabrication

Abstract The main goal in biofabrication approach is to build living tissue substitutes on demand. In order to create functional tissue structures, additive manufacturing (AM) technologies are being increasingly considered. They allow generating functional structures created out of CAD models within a short period of time and with a very high precision. Different techniques are already established to build three-dimensional (3D) complex cell-loaded structures. One of these robotic additive fabrication techniques is the ink jet technology which is highly promising for biofabrication. This technique allows to process very small amounts of liquids or low-viscous polymer solutions e.g. to set biomolecules and cells in a suitable structure. The aim of this study is to evaluate a piezo inkjet printing device which is integrated in a commercial modular instrument platform together with a bioplotting system for biofabrication. The inkjet device is able to print single ink droplets of different volumes by controlling the applied voltage and the number of drops released to the spot. In this work different selective sets of parameters influencing the droplet formation and the spot size have been investigated. It has been proven that inkjet printing process in combination with fibrin hydrogel and bone marrow stromal cells is cytocompatible. In summary, the applied piezo inkjet printing is shown to be completely programmable, accurate and the resolution of the device allowed printing of various patterns with biomaterials and vital cells.

Fabrication of Cell-Loaded Two-Phase 3D Constructs for Tissue Engineering

Hydrogel optimisation for biofabrication considering shape stability/mechanical properties and cell response is challenging. One approach to tackle this issue is to combine different additive manufacturing techniques, e.g., hot-melt extruded thermoplastics together with bioplotted cell loaded hydrogels in a sequential plotting process. This method enables the fabrication of 3D constructs mechanically supported by the thermoplastic structure and biologically functionalised by the hydrogel phase. In this study, polycaprolactone (PCL) and polyethylene glycol (PEG) blend (PCL-PEG) together with alginate dialdehyde gelatine hydrogel (ADA-GEL) loaded with stromal cell line (ST2) were investigated. PCL-PEG blends were evaluated concerning plotting properties to fabricate 3D scaffolds, namely miscibility, wetting behaviour and in terms of cell response. Scaffolds were characterised considering pore size, porosity, strut width, degradation behaviour and mechanical stability. Blends showed improved hydrophilicity and cell response with PEG blending increasing the degradation and decreasing the mechanical properties of the scaffolds. Hybrid constructs with PCL-PEG blend and ADA-GEL were fabricated. Cell viability, distribution, morphology and interaction of cells with the support structure were analysed. Increased degradation of the thermoplastic support structure and proliferation of the cells not only in the hydrogel, but also on the thermoplastic phase, indicates the potential of this novel material combination for biofabricating 3D tissue engineering scaffolds.