Characterization of self-healing hydrogels for biomedical applications

Self-healing hydrogels have become attractive biomaterials due to their ability to repair their initial structure and properties in response to damage. When designing ideal self-healing hydrogels the understanding of their properties but also the actual healing process is required. Even though there currently are different characterization methods used, the lack of standardization makes comparison of different hydrogels difficult. The challenges in standardization arise, for example, from the use of different healing methods (i.e. healing environments) or different testing equipments used. In order to help the comparison of hydrogels, a group of characterization methods should be chosen and the measuring parameters and results in the literature should be presented more consistently. The characterization should include methods suitable to determine the presence of reversible interactions and their reversibility study, to investigate the self-healability of hydrogels and to determine the healing efficiencies of hydrogels, not forgetting time dependence and dynamics of self-healing. More quantitative, as well as theoretical studies are recommended. In this review different general characterization methods, including different measuring parameters and environments, used for self-healing hydrogels are charted, but also additional methods suitable for injectable/3D-bioprintable and conductive self-healing hydrogels are discussed. Some challenges of each method and future aspects for self-healing hydrogels and their characterization are also given.publishedVersionPeer reviewe

Design aspects and characterization of hydrogel-based bioinks for extrusion-based bioprinting

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Injectable and self-healing biobased composite hydrogels as future anticancer therapeutic biomaterials

Abstract Self-healing composite hydrogels are prepared from sustainable biopolymers by a green chemistry approach and analyzed by physicochemical and mechanical characterization techniques for future injectable anticancer biomaterials. Water-soluble chitosan (WSC) was prepared by grafting polyethylene glycol (PEG), glutamic acid and gallic acid onto the chitosan chain by carbodiimide chemistry. This WSC showed fast gelation (t ≈ < 60 seconds) with benzaldehyde-terminated 4-arm-PEG as a crosslinker through an amine/aldehyde Schiff base reaction. The compression modulus of these gels can be controlled between 6 and 67 kPa, which was dependent on both the crosslinker content as well as the total solid content (T%). It showed injectability and complete self-healing ability at the lower solid content (T = 2%). The hydrogel nanocomposites (HNCs) were synthesized together with gold (Au) and silver (Ag) nanoparticles (NPs) and tested for cytotoxicity using fibroblast cells (WI-38) for 48 hours, which showed good biocompatibility. The in-vitro assay against cancer cells (U87MG) for 48 hours indicated that only the HNCs with incorporated AuNPs were effective agents for cancer cell apoptosis in contrast to pristine gel, pure NPs (Ag and AuNPs) and HCNs with AgNPs. Therefore, these HNCs could be effective chemotherapeutic materials for designing anticancer nanomedicines in the future.publishedVersionPeer reviewe

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.publishedVersionPeer reviewe

Monitoring the gelation of gellan gum with torsion rheometry and brightness-mode ultrasound

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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.publishedVersionPeer reviewe

Optical projection tomography technique for image texture and mass transport studies in hydrogels based on gellan gum

The microstructure and permeability are crucial factors for the development of hydrogels for tissue engineering, since they influence cell nutrition, penetration and proliferation. The currently available imaging methods able to characterize hydrogels have many limitations. They often require sample drying and other destructive processing, which can change hydrogel structure, or they have limited imaging penetration depth. In this work, we show for the first time an alternative non-destructive method, based on optical projection tomography (OPT) imaging, to characterize hydrated hydrogels without the need of sample processing. As proof of concept we used gellan gum (GG) hydrogels obtained by several crosslinking methods. Transmission mode OPT was used to analyse image microtextures and emission mode OPT to study mass transport. Differences in hydrogels structure related to different types of crosslinking and between modified and native GG were found through the acquired Haralickâ s image texture features followed by multiple discriminant analysis (MDA). In mass transport studies, the mobility of FITC-dextran (MW 20, 150, 2000 kDa) was analysed through the macroscopic hydrogel. The FITC-dextran velocities were found to be inversely proportional to the size of the dextran as expected. Furthermore, the threshold size in which the transport is affected by the hydrogel mesh was found to be 150 kDa (Stokesâ radii between 69 à and 95 à ). On the other hand, the mass transport study allowed us to define an index of homogeneity to assess the crosslinking distribution, structure inside the hydrogel and repeatability of hydrogel production. As a conclusion,  we showed that  the set of OPT imaging based material characterization methods presented here are useful for screening many characteristics of hydrogel compositions in relatively short time in an inexpensive manner, providing  tools for improving the process of designing hydrogels for tissue engineering and drugs/cells delivery applications.Portuguese Foundation for Science and Technology (FCT) - Grant No. SFRH/BPD/100590/201

Evaluation of the sterilization effect on biphasic scaffold based on bioactive glass and polymer honeycomb membrane

The sterilization is a core preoccupation when it comes to implantable biomaterials. The most common in industry is the gamma sterilization; however, the radiation used in this method can induce modifications in the material properties. This study investigates the impact of such radiations on the physicochemical properties and biological toxicity of a new biomaterial based on a poly-l-co-d,l-lactide polymer honeycomb membrane and bioactive glass (BG), combined, to form an assembly (membrane/BG assembly). The investigated BGs are the S53P4, which is FDA approved and clinically used, and 13-93B20, a BG containing boron promising for bone regeneration. Infrared and photoluminescence measurements revealed that, upon irradiation, defects are created in the BGs molecular matrix. Defects were identified to be mainly non-bridging oxygen hole center and occur in higher proportion in the 13-93B20 making it more sensitive to irradiation compared to the S53P4. However, the irradiation does not significantly impact the structure of the BGs. On the membrane side, the molecular weight is divided by two resulting in a lower shear stress resistance. However, the membrane honeycomb topography does not seem to be impacted by the irradiation. In contact with cells, no toxicity effect was observed, and BGs keep their bioactive properties by releasing ions beneficial to the cell fate and with no influence on apatite precipitation speed. Overall, this study showed that, despite some impact on the physicochemical properties, the irradiation does not induce deleterious effect on the membrane/BG assemblies and is therefore a suitable method for the sterilization of this novel biomaterial.Peer reviewe