Functionalized Gelatin/Polysaccharide Hydrogels for Encapsulation of Hepatocytes.

Liver diseases represent a considerable burden to patients and healthcare systems. Hydrogels play an important role in the engineering of soft tissues and may be useful for embedding hepatocytes for different therapeutic interventions or the development of in vitro models to study the pathogenesis of liver diseases or testing of drugs. Here, we developed two types of hydrogels by crosslinking hydrazide-functionalized gelatin with either oxidized dialdehyde hyaluronan or alginate through the formation of hydrazone bonds. Gel formulations were studied through texture analysis and rheometry, showing mechanical properties comparable to those of liver tissue while also demonstrating long-term stability. The biocompatibility of hydrogels and their ability to host hepatocytes was studied in vitro in comparison to pure gelatin hydrogels crosslinked by transglutaminase using the hepatocellular line HepG2. It was found that HepG2 cells could be successfully embedded in the hydrogels, showing no signs of gel toxicity and proliferating in a 3D environment comparable to pure transglutaminase cross-linked gelatin hydrogels used as control. Altogether, hydrazide gelatin in combination with oxidized polysaccharides makes stable in situ gelling systems for the incorporation of hepatocytes, which may pave the way for use in liver tissue engineering and drug testing.

3D printed neural tissues with in situ optical dopamine sensors.

Engineered neural tissues serve as models for studying neurological conditions and drug screening. Besides observing the cellular physiological properties, in situ monitoring of neurochemical concentrations with cellular spatial resolution in such neural tissues can provide additional valuable insights in models of disease and drug efficacy. In this work, we demonstrate the first three-dimensional (3D) tissue cultures with embedded optical dopamine (DA) sensors. We developed an alginate/Pluronic F127 based bio-ink for human dopaminergic brain tissue printing with tetrapodal-shaped-ZnO microparticles (t-ZnO) additive as the DA sensor. DA quenches the autofluorescence of t-ZnO in physiological environments, and the reduction of the fluorescence intensity serves as an indicator of the DA concentration. The neurons that were 3D printed with the t-ZnO showed good viability, and extensive 3D neural networks were formed within one week after printing. The t-ZnO could sense DA in the 3D printed neural network with a detection limit of 0.137 µM. The results are a first step toward integrating tissue engineering with intensiometric biosensing for advanced artificial tissue/organ monitoring.

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

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

Metal Ion Doping of Alginate-Based Surface Coatings Induces Adipogenesis of Stem Cells.

Metal ions are important effectors of protein and cell functions. Here, polyelectrolyte multilayers (PEMs) made of chitosan (Chi) and alginate (Alg) were doped with different metal ions (Ca2+, Co2+, Cu2+, and Fe3+), which can form bonds with their functional groups. Ca2+ and Fe3+ ions can be deposited in PEM at higher quantities resulting in more positive ζ potentials and also higher water contact angles in the case of Fe3+. An interesting finding was that the exposure of PEM to metal ions decreases the elastic modulus of PEM. Fourier transformed infrared (FTIR) spectroscopy of multilayers provides evidence of interaction of metal ions with the carboxylic groups of Alg but not for hydroxyl and amino groups. The observed changes in wetting and surface potential are partly related to the increased adhesion and proliferation of multipotent C3H10T1/2 fibroblasts in contrast to plain nonadhesive [Chi/Alg] multilayers. Specifically, PEMs doped with Cu2+ and Fe3+ ions greatly promote cell attachment and adipogenic differentiation, which indicates that changes in not only surface properties but also the bioactivity of metal ions play an important role. In conclusion, metal ion-doped multilayer coatings made of alginate and chitosan can promote the differentiation of multipotent cells on implants without the use of other morphogens like growth factors.

Control of Blood Coagulation by Hemocompatible Material Surfaces-A Review.

Hemocompatibility of biomaterials in contact with the blood of patients is a prerequisite for the short- and long-term applications of medical devices such as cardiovascular stents, artificial heart valves, ventricular assist devices, catheters, blood linings and extracorporeal devices such as artificial kidneys (hemodialysis), extracorporeal membrane oxygenation (ECMO) and cardiopulmonary bypass. Although lower blood compatibility of materials and devices can be handled with systemic anticoagulation, its side effects, such as an increased bleeding risk, make materials that have a better hemocompatibility highly desirable, particularly in long-term applications. This review provides a short overview on the basic mechanisms of blood coagulation including plasmatic coagulation and blood platelets, as well as the activation of the complement system. Furthermore, a survey on concepts for tailoring the blood response of biomaterials to improve the hemocompatibility of medical devices is given which covers different approaches that either inhibit interaction of material surfaces with blood components completely or control the response of the coagulation system, blood platelets and leukocytes.

Hydrogels Based on Oxidized Cellulose Sulfates and Carboxymethyl Chitosan: Studies on Intrinsic Gel Properties, Stability, and Biocompatibility.

Cellulose and chitosan are excellent components for the fabrication of bioactive scaffolds, as they are biocompatible and abundantly available. Their derivatives Ocarboxymethyl chitosan (CMChi) and oxidized cellulose sulfate (oxCS) can form in situ gelling, bioactive hydrogels, due to the formation of imine bonds for crosslinking. Here the influence of the degrees of sulfation (DS), oxidation (DO), and the molecular weight of oxCS on intrinsic and rheological properties of such hydrogels and their ability to support the survival and growth of human-adipose-derived stem cells (hADSC) is investigated. It is found that the pH of the hydrogels is generally slightly acidic, while their network density and E-modulus are found to be dependent on the DS and DO, which makes the properties of hydrogels tunable. Extensive studies show that hydrogels can be stable for up to 14 days and that their stability is largely dependent on the DO, molecular weight, and the components mixing ratio. Cytotoxicity studies of the hydrogel with hADSCs show biocompatible gels in dependence on the molecular weight and degree of oxidation with viable cells up to 14 days. These findings can help to develop specifically tailored hydrogels for tissue engineering applications to replace different types of connective tissue.

Arteriovenous access in hemodialysis: A multidisciplinary perspective for future solutions.

In hemodialysis, vascular access is a key issue. The preferred access is an arteriovenous fistula on the non-dominant lower arm. If the natural vessels are insufficient for such access, the insertion of a synthetic vascular graft between artery and vein is an option to construct an arteriovenous shunt for punctures. In emergency situations and especially in elderly with narrow and atherosclerotic vessels, a cuffed double-lumen catheter is placed in a larger vein for chronic use. The latter option constitutes a greater risk for infections while arteriovenous fistula and arteriovenous shunt can fail due to stenosis, thrombosis, or infections. This review will recapitulate the vast and interdisciplinary scenario that characterizes hemodialysis vascular access creation and function, since adequate access management must be based on knowledge of the state of the art and on future perspectives. We also discuss recent developments to improve arteriovenous fistula creation and patency, the blood compatibility of arteriovenous shunt, needs to avoid infections, and potential development of tissue engineering applications in hemodialysis vascular access. The ultimate goal is to spread more knowledge in a critical area of medicine that is importantly affecting medical costs of renal replacement therapies and patients' quality of life.

Thermoresponsive polymers and their biomedical application in tissue engineering - a review.

Thermoresponsive polymers hold great potential in the biomedical field, since they enable the fabrication of cell sheets, in situ drug delivery and 3D-printing under physiological conditions. In this review we provide an overview of several thermoresponsive polymers and their application, with focus on poly(N-isopropylacrylamide)-surfaces for cell sheet engineering. Basic knowledge of important processes like protein adsorption on surfaces and cell adhesion is provided. For different thermoresponsive polymers, namely PNIPAm, Pluronics, elastin-like polypeptides (ELP) and poly(N-vinylcaprolactam) (PNVCL), synthesis and basic chemical and physical properties have been described and the mechanism of their thermoresponsive behavior highlighted. Fabrication methods of thermoresponsive surfaces have been discussed, focusing on PNIPAm, and describing several methods in detail. The latter part of this review is dedicated to the application of the thermoresponsive polymers and with regard to cell sheet engineering, the process of temperature-dependent cell sheet detachment is explained. We provide insight into several applications of PNIPAm surfaces in cell sheet engineering. For Pluronics, ELP and PNVCL we show their application in the field of drug delivery and tissue engineering. We conclude, that research of thermoresponsive polymers has made big progress in recent years, especially for PNIPAm since the 1990s. However, manifold research possibilities, e.g. in surface fabrication and 3D-printing and further translational applications are conceivable in near future.

Synthesis and Characterization of Oxidized Polysaccharides for In Situ Forming Hydrogels.

Polysaccharides are widely used as building blocks of scaffolds and hydrogels in tissue engineering, which may require their chemical modification to permit crosslinking. The goal of this study was to generate a library of oxidized alginate (oALG) and oxidized hyaluronic acid (oHA) that can be used for in situ gelling hydrogels by covalent reaction between aldehyde groups of the oxidized polysaccharides (oPS) and amino groups of carboxymethyl chitosan (CMC) through imine bond formation. Here, we studied the effect of sodium periodate concentration and reaction time on aldehyde content, molecular weight of derivatives and cytotoxicity of oPS towards 3T3-L1 fibroblasts. It was found that the molecular weights of all oPs decreased with oxidation and that the degree of oxidation was generally higher in oHA than in oALG. Studies showed that only oPs with an oxidation degree above 25% were cytotoxic. Initial studies were also done on the crosslinking of oPs with CMC showing with rheometry that rather soft gels were formed from higher oxidized oPs possessing a moderate cytotoxicity. The results of this study indicate the potential of oALG and oHA for use as in situ gelling hydrogels or inks in bioprinting for application in tissue engineering and controlled release.

Effect of Different Crosslinking Strategies on Physical Properties and Biocompatibility of Freestanding Multilayer Films Made of Alginate and Chitosan.

Freestanding multilayer films prepared by layer-by-layer technique have attracted interest as promising materials for wound dressings. The goal is to fabricate freestanding films using chitosan (CHI) and alginate (ALG) including subsequent crosslinking to improve the mechanical properties of films while maintaining their biocompatibility. Three crosslinking strategies are investigated, namely use of calcium ions for crosslinking ALG, 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide combined with N-hydroxysuccinimide for crosslinking ALG with CHI, and Genipin for crosslinking chitosan inside the films. Different characteristics, such as surface morphology, wettability, swelling, roughness, and mechanical properties are investigated showing that films became thinner, exhibited rougher surfaces, had lower water uptake, and increased mechanical strength after crosslinking. Changes of wettability are moderate and dependent on the crosslinking method. In vitro cytotoxicity and cell attachment studies with human dermal fibroblasts show that freestanding CHI-ALG films represent a poorly adhesive substratum for fibroblasts, while studies using incubation of plastic-adherent fibroblast beneath floating films show no signs of cytotoxicity in a time frame of 7 days. Results from cell experiments combined with film characteristics after crosslinking, indicate that crosslinked freestanding films made of ALG and CHI may be interesting candidates for wound dressings.