Fabrication of Hydrogel-Based Composite Fibers and Computer Simulation of the Filler Dynamics in the Composite Flow.

Fibrous structures with anisotropic fillers as composites have found increasing interest in the field of biofabrication since they can mimic the extracellular matrix of anisotropic tissues such as skeletal muscle or nerve tissue. In the present work, the inclusion of anisotropic fillers in hydrogel-based filaments with an interpenetrating polymeric network (IPN) was evaluated and the dynamics of such fillers in the composite flow were analyzed using computational simulations. In the experimental part, microfabricated rods (200 and 400 µm length, 50 µm width) were used as anisotropic fillers in extrusion of composite filaments using two techniques of wet spinning and 3D printing. Hydrogels such as oxidized alginate (ADA) and methacrylated gelatin (GelMA) were used as matrices. In the computational simulation, a combination of computational fluid dynamics and coarse-grained molecular dynamics was used to study the dynamics of rod-like fillers in the flow field of a syringe. It showed that, during the extrusion process, microrods are far from being well aligned. Instead, many of them tumble on their way through the needle leading to a random orientation in the fiber which was confirmed experimentally.

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.

Topical systems for the controlled release of antineoplastic Drugs: Oxidized Alginate-Gelatin Hydrogel/Unilamellar vesicles.

The efficacy of chemotherapeutic procedures relies on delivering proper concentrations of anti-cancer drugs in the tumor surroundings, so as to prevent potential side effects on healthy tissues. Novel drug carrier platforms should not just be able to deliver anticancer molecules, but also allow for adjustements in the way these drugs are administered to the patients. We developed a system for delivering water-insoluble drugs, based on the use of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), or bis(2-ethylhexyl) sulfosuccinate benzyl-n-hexadecyldimethylammonium (BHD-AOT), embedded into oxidized alginate-gelatin (ADA/Gel) hydrogel, emulating a patch for topic applications. After being loaded with curcumin, cancer cells such as human colorectal adenocarcinoma (HCT116 and DLD-1) and melanoma cell lines (MEL501), and non-malignant cells such as mammary epithelial cell lines (NMuMG) and embryonal fibroblasts (NIH 3T3 or NEO cells) were analyzed for biocompatibility and cytotoxic effects. The results show that the proposed system can load comparatively higher concentrations of the drug (with respect to other nano/microcarriers in the literature), and that it can enhance the likelihood of the drug being uptaken by cancer cells instead of non-malignant cells. These assays were complemented by diffusion studies across the stratum corneum of rat skin, with the aim of determining the system's efficiency during topical application. Finally, the stability of the patch was tested after lyophilization to determine its potential pharmaceutical use. As a whole, the combined system represents a highly reliable and robust method for embedding and delivering complex insoluble chemotherapeutical molecules, and it is less invasive than other alternative methods in the literature.

3D Bioprinting of Multifunctional Dynamic Nanocomposite Bioinks Incorporating Cu-Doped Mesoporous Bioactive Glass Nanoparticles for Bone Tissue Engineering.

Bioprinting has seen significant progress in recent years for the fabrication of bionic tissues with high complexity. However, it remains challenging to develop cell-laden bioinks exhibiting superior physiochemical properties and bio-functionality. In this study, a multifunctional nanocomposite bioink is developed based on amine-functionalized copper (Cu)-doped mesoporous bioactive glass nanoparticles (ACuMBGNs) and a hydrogel formulation relying on dynamic covalent chemistry composed of alginate dialdehyde (oxidized alginate) and gelatin, with favorable rheological properties, improved shape fidelity, and structural stability for extrusion-based bioprinting. The reversible dynamic microenvironment in combination with the impact of cell-adhesive ligands introduced by aminated particles enables the rapid spreading (within 3 days) and high survival (>90%) of embedded human osteosarcoma cells and immortalized mouse bone marrow-derived stroma cells. Osteogenic differentiation of primary mouse bone marrow stromal stem cells (BMSCs) and angiogenesis are promoted in the bioprinted alginate dialdehyde-gelatin (ADA-GEL or AG)-ACuMBGN scaffolds without additional growth factors in vitro, which is likely due to ion stimulation from the incorporated nanoparticles and possibly due to cell mechanosensing in the dynamic matrix. In conclusion, it is envisioned that these nanocomposite bioinks can serve as promising platforms for bioprinting complex 3D matrix environments providing superior physiochemical and biological performance for bone tissue engineering.

Potential of Laponite® incorporated oxidized alginate-gelatin (ADA-GEL) composite hydrogels for extrusion-based 3D printing.

The concept of adding inorganic fillers into hydrogels to form hydrogel nanocomposites often provides advantageous properties which can be exploited for successful 3D biofabrication. In this study, a new composite hydrogel combining oxidized alginate-gelatin (ADA-GEL) hydrogel and Laponite® nanoclay as inorganic nanofiller was successfully developed and characterized. The results showed that the addition of 0.5% (wt/vol) Laponite® nanoplatelets improved the printability of ADA-GEL hydrogels enabling the fabrication of detailed structures since a low effect of material spreading and reduced tendency to pore closure appeared. Furthermore, a comparison of different needle types (cylindrical and conical; same inner diameter of 250 µm) in filament fusion test showed that the pattern dispensed by cylindrical tip has enhanced printing accuracy and pattern fidelity when compared with the pattern from conical tip. A glass flip test determined a processing window of 1-2 h after composite ink preparation. Overall, Laponite® /ADA-GEL hydrogel composites are confirmed as promising inks for 3D bioprinting.

3D printing of bioreactors in tissue engineering: A generalised approach.

3D printing is a rapidly evolving field for biological (bioprinting) and non-biological applications. Due to a high degree of freedom for geometrical parameters in 3D printing, prototype printing of bioreactors is a promising approach in the field of Tissue Engineering. The variety of printers, materials, printing parameters and device settings is difficult to overview both for beginners as well as for most professionals. In order to address this problem, we designed a guidance including test bodies to elucidate the real printing performance for a given printer system. Therefore, performance parameters such as accuracy or mechanical stability of the test bodies are systematically analysed. Moreover, post processing steps such as sterilisation or cleaning are considered in the test procedure. The guidance presented here is also applicable to optimise the printer settings for a given printer device. As proof of concept, we compared fused filament fabrication, stereolithography and selective laser sintering as the three most used printing methods. We determined fused filament fabrication printing as the most economical solution, while stereolithography is most accurate and features the highest surface quality. Finally, we tested the applicability of our guidance by identifying a printer solution to manufacture a complex bioreactor for a perfused tissue construct. Due to its design, the manufacture via subtractive mechanical methods would be 21-fold more expensive than additive manufacturing and therefore, would result in three times the number of parts to be assembled subsequently. Using this bioreactor we showed a successful 14-day-culture of a biofabricated collagen-based tissue construct containing human dermal fibroblasts as the stromal part and a perfusable central channel with human microvascular endothelial cells. Our study indicates how the full potential of biofabrication can be exploited, as most printed tissues exhibit individual shapes and require storage under physiological conditions, after the bioprinting process.

Advancing bioinks for 3D bioprinting using reactive fillers: A review.

The growing demand for personalized implants and tissue scaffolds requires advanced biomaterials and processing strategies for the fabrication of three-dimensional (3D) structures mimicking the complexity of the extracellular matrix. During the last years, biofabrication approaches like 3D printing of cell-laden (soft) hydrogels have been gaining increasing attention to design such 3D functional environments which resemble natural tissues (and organs). However, often these polymeric hydrogels show poor stability and low printing fidelity and hence various approaches in terms of multi-material mixtures are being developed to enhance pre- and post-printing features as well as cytocompatibility and post-printing cellular development. Additionally, bioactive properties improve the binding to the surrounding (host) tissue at the implantation site. In this review we focus on the state-of-the-art of a particular type of heterogeneous bioinks, which are composed of polymeric hydrogels incorporating inorganic bioactive fillers. Such systems include isotropic and anisotropic silicates like bioactive glasses and nanoclays or calcium-phosphates like hydroxyapatite (HAp), which provide in-situ crosslinking effects and add extra functionality to the matrix, for example mineralization capability. The present review paper discusses in detail such bioactive composite bioink systems based on the available literature, revealing that a great variety has been developed with substantially improved bioprinting characteristics, in comparison to the pure hydrogel counterparts, and enabling high viability of printed cells. The analysis of the results of the published studies demonstrates that bioactive fillers are a promising addition to hydrogels to print stable 3D constructs for regeneration of tissues. Progress and challenges of the development and applications of such composite bioink approaches are discussed and avenues for future research in the field are presented. STATEMENT OF SIGNIFICANCE: Biofabrication, involving the processing of biocompatible hydrogels including cells (bioinks), is being increasingly applied for developing complex tissue and organ mimicking structures. A variety of multi-material bioinks is being investigated to bioprint 3D constructs showing shape stability and long-term biological performance. Composite hydrogel bioinks incorporating inorganic bioreactive fillers for 3D bioprinting are the subject of this review paper. Results reported in the literature highlight the effect of bioactive fillers on bioink properties, printability and on cell behavior during and after printing and provide important information for optimizing the design of future bioinks for biofabrication, exploiting the extra functionalities provided by inorganic fillers. Further functionalization with drugs/growth factors can target enhanced printability and local drug release for more specialized biomedical therapies.

Ionically and Enzymatically Dual Cross-Linked Oxidized Alginate Gelatin Hydrogels with Tunable Stiffness and Degradation Behavior for Tissue Engineering.

Hydrogels that allow for the successful long-term in vitro culture of cell-biomaterial systems to enable the maturation of tissue engineering constructs are highly relevant in regenerative medicine. Naturally derived polysaccharide-based hydrogels promise to be one material group with enough versatility and chemical functionalization capability to tackle the challenges associated with long-term cell culture. We report a marine derived oxidized alginate, alginate dialdehyde (ADA), and gelatin (GEL) system (ADA-GEL), which is cross-linked via ionic (Ca2+) and enzymatic (microbial transglutaminase, mTG) interaction to form dually cross-linked hydrogels. The cross-linking approach allowed us to tailor the stiffness of the hydrogels in a wide range (from <5 to 120 kPa), without altering the initial ADA and GEL hydrogel chemistry. It was possible to control the degradation behavior of the hydrogels to be stable for up to 30 days of incubation. Increasing concentrations of mTG cross-linker solutions allowed us to tune the degradation behavior of the ADA-GEL hydrogels from fast (<7 days) to moderate (14 days) and slow (>30 days) degradation kinetics. The cytocompatibility of mTG cross-linked ADA-GEL was assessed using NIH-3T3 fibroblasts and ATDC-5 mouse teratocarcinoma cells. Both cell types showed highly increased cellular attachment on mTG cross-linked ADA-GEL in comparison to Ca2+ cross-linked hydrogels. In addition, ATDC-5 cells showed a higher proliferation on mTG cross-linked ADA-GEL hydrogels in comparison to tissue culture polystyrene control substrates. Further, the attachment of human umbilical vein endothelial cells (HUVEC) on ADA-GEL (+) mTG was confirmed, proving the suitability of mTG+Ca2+ cross-linked ADA-GEL for several cell types. Summarizing, a promising platform to control the properties of ADA-GEL hydrogels is presented, with the potential to be applied in long-term cell culture investigations such as cartilage, bone, and blood-vessel engineering, as well as for biofabrication.

Synthesis and Characterization of Tissue Plasminogen Activator-Functionalized Superparamagnetic Iron Oxide Nanoparticles for Targeted Fibrin Clot Dissolution.

Superparamagnetic iron oxide nanoparticles (SPIONs) have attracted great attention in many biomedical fields and are used in preclinical/experimental drug delivery, hyperthermia and medical imaging. In this study, biocompatible magnetite drug carriers, stabilized by a dextran shell, were developed to carry tissue plasminogen activator (tPA) for targeted thrombolysis under an external magnetic field. Different concentrations of active tPA were immobilized on carboxylated nanoparticles through carbodiimide-mediated amide bond formation. Evidence for successful functionalization of SPIONs with carboxyl groups was shown by Fourier transform infrared spectroscopy. Surface properties after tPA immobilization were altered as demonstrated by dynamic light scattering and ζ potential measurements. The enzyme activity of SPION-bound tPA was determined by digestion of fibrin-containing agarose gels and corresponded to about 74% of free tPA activity. Particles were stored for three weeks before a slight decrease in activity was observed. tPA-loaded SPIONs were navigated into thrombus-mimicking gels by external magnets, proving effective drug targeting without losing the protein. Furthermore, all synthesized types of nanoparticles were well tolerated in cell culture experiments with human umbilical vein endothelial cells, indicating their potential utility for future therapeutic applications in thromboembolic diseases.