Evaluation of Various Types of Alginate Inks for Light-Mediated Extrusion 3D Printing.

Naturally derived biopolymers modifying or combining with other components are excellent candidates to promote the full potential of additive manufacturing in biomedicine, cosmetics, and the food industry. This work aims to develop new photo-cross-linkable alginate-based inks for extrusion 3D printing. Specifically, this work is focused on the effect of the addition of cross-linkers with different chemical structures (polyethylene glycol diacrylate (PEGDA), N,N'-methylenebisacrylamide (NMBA), and acrylic acid (AA)) in the potential printability and physical properties of methacrylated alginate (AlgMe) hydrogels. Although all inks showed maximum photo-curing conversions and gelation times less than 2 min, only those structures printed with the inks incorporating cross-linking agents with flexible and long chain structure (PEGDA and AA) displayed acceptable size accuracy (~0.4-0.5) and printing index (Pr ~1.00). The addition of these cross-linking agents leads to higher Young's moduli (from 1.6 to 2.0-2.6 KPa) in the hydrogels, and their different chemical structures results in variations in their mechanical and rheological properties. However, similar swelling ability (~15 swelling factor), degradability (~45 days 100% weight loss), and cytocompatibility (~100%) were assessed in all the systems, which is of great importance for the final applicability of these hydrogels.

Extrusion-Based 3D Printing of Photocrosslinkable Chitosan Inks.

Photocuring of chitosan has shown great promise in the extrusion-based 3D printing of scaffolds for advanced biomedical and tissue engineering applications. However, the poor mechanical stability of methacrylated chitosan photocuring ink restricts its applicability. The inclusion of co-networks by means of simultaneous polycomplex formation is an effective method by which to solve this drawback, but the formed hydrogel inks are not printable. This work aims to develop new photocurable chitosan inks based on the simultaneous photocrosslinking of methacrylated chitosan (CHIMe) with N,N'-methylenebisacrylamide, polyethylene glycol diacrylate, and acrylic acid to be applied in extrusion 3D printing. Interestingly, the polycomplex co-network corresponding to the acrylic-acid-based ink could be successfully printed by the here-presented simultaneous photocuring strategy. Further, the conversion of photocrosslinking was studied via photo-DSC analyses that revealed a clear dependence on the chemical structure of the employed crosslinking agents (from 40 to ~100%). In addition, the mechanical and rheological properties of the photocured hydrogels were comparatively studied, as well as the printing quality of the extruded scaffolds. The newly developed chitosan photocurable inks demonstrated extrusion printability (squareness ~0.90; uniformity factor ~0.95) and tunable mechanical properties (Young modulus 14-1068 Pa) by means of different crosslinking approaches according to the chemical architecture of the reactive molecules employed. This work shows the great potential of photocrosslinkable chitosan inks.

Multifunctional antibacterial chitosan-based hydrogel coatings on Ti6Al4V biomaterial for biomedical implant applications.

Among biomedical community, great efforts have been realized to develop antibacterial coatings that avoid implant-associated infections. To date, conventional mono-functional antibacterial strategies have not been effective enough for successful long-term implantations. Consequently, researchers have recently focused their attention on novel bifunctional or multifunctional antibacterial coatings, in which two or more antibacterial mechanisms interact synergistically. Thus, in this work different chitosan-based (CHI) hydrogel coatings were created on Ti6Al4V surface using genipin (Ti-CHIGP) and polyethylene glycol (Ti-CHIPEG) crosslinking agents. Hydrogel coatings demonstrated an exceptional in vivo biocompatibility plus a remarkable ability to promote cell proliferation and differentiation. Lastly, hydrogel coatings demonstrated an outstanding bacteria-repelling (17-28 % of S. aureus and 33-43 % of E. coli repelled) and contact killing (186-222 % of S. aureus and 72-83 % of E. coli damaged) ability. Such bifunctional antibacterial activity could be further improved by the controlled release of drugs resulting in powerful multifunctional antibacterial coatings.

Covalent Cross-Linking as a Strategy to Prepare Water-Dispersible Chitosan Nanogels.

Due to the environmental problems generated by petroleum derivative polymers as mentioned in Agenda 2030, the use of natural polymers is increasing. Among them, cellulose and chitin are the most widespread biopolymers available in nature. Chitosan, obtained from chitin, is a really good candidate to develop nanocarriers due to its polyelectrolyte nature and ease of chemical modification. However, chitosan presents a solubility drawback in an aqueous medium at physiological pH (pH = 7.4), which restricts its applicability in biomedicine. In this work, nanogels were successfully synthesized from chitosan systems with different water solubilities (chitosan, oligosaccharide chitosan, and quaternized chitosan) using the reverse microemulsion method and polyethylene glycol diacid (PEGBCOOH) as a covalent cross-linking agent. Cross-linking with PEGBCOOH was analyzed by proton nuclear magnetic resonance (1H-NMR), which allowed for nanogels to be prepared whose size and swelling were comparatively studied by transmission electron microscopy (TEM) and dynamic light scattering (DLS) and zeta potential, respectively. The particle size of the swollen nanogels showed a different pH-responsive behavior that decreased for chitosan, increased for oligosaccharide chitosan, and remained constant for quaternized chitosan. Nevertheless, a drastic reduction was observed in all cases in the culture medium. Along the same line, the dispersibility of the synthesized nanogels in different media was comparatively evaluated, showing similar values for the nanogels prepared from soluble chitosans than for water insoluble chitosan as a consequence of the cross-linking with PEGBCOOH. After 6 months of storage of the dried nanogels, the water dispersibility values remained constant in all cases, demonstrating the stabilizing effect of the employed cross-linking agent and the potential use of synthesized nanogels as substrates for drug delivery.

Hyaluronic acid-based hydrogel coatings on Ti6Al4V implantable biomaterial with multifunctional antibacterial activity.

Today, the treatment of implant-associated infections with conventional mono-functional antibacterial coatings has not been effective enough for a prosperous long-term implantation. Therefore, biomedical industry is making considerable efforts on the development of novel antibacterial coatings with a combination of more than one antibacterial strategies that interact synergistically to reinforce each other. Therefore, in this work hyaluronic acid-based (HA) hydrogel coatings were created on the surface Ti6Al4V biomaterial with 1,4-butanediol diglycidyl ether (Ti-HABDDE) and divinyl sulfone (Ti-HADVS) crosslinking agents. Hydrogel coatings displayed an extraordinary in vivo biocompatibility, a remarkable ability to promote cell proliferation, differentiation and mineralization, and capability to sustainedly release drugs. Finally, HA-based hydrogel coatings demonstrated an outstanding multifunctional antibacterial activity: bacteria-repelling (51-55 % of S. aureus and 27-40 % of E. coli), bacteria-killing (82-119 % of S. aureus and 83-87 % of E. coli) and bactericide release killing (drug-loaded hydrogel coatings, R > 2).

Graphene-Enhanced Methacrylated Alginate Gel Films for Sustainable Dye Removal in Water Purification.

Self-standing nanocomposite films were prepared by three-dimensional UV-induced radical copolymerization of methacrylated alginate (MALG) with acrylic acid (AA) and reinforced with graphene oxide (GO) to improve both mechanical strength and dye adsorption capacity in wastewater decontamination operations. Dynamic mechanical-thermal analysis revealed variations in storage modulus: the higher the GO content, the higher the storage modulus (E') values. Also, the higher the temperature (associated with a lower and lower water content of films), the larger values of E' for the films of the same composition (E'(25 °C) = 676.6-1538.7 MPa; E'(100 °C) = 886.9-2066.6 MPa), providing insights into the compatibility between GO and the MALG/AA matrix, as well as, assessing the improvement in the nanocomposite's final mechanical properties. These crosslinked films in a dry state exhibited rapid water uptake and relatively short drying times (ca. 30 min at room temperature for the MALG/AA/GO composites) resulting from the swelling-drying studies and water contact angle measurements. The efficacy of methylene blue removal from water assessed via UV-VIS spectrometry revealed excellent results, expressed as an adsorption yield of 70-80% and 85-98% after 30 h and 258 h, respectively, of immersion time of films into an MB aqueous solution of 12.5 mg/L (as the contaminated water model). The reusability of the same films was evaluated by consecutive extraction processes of MB from the composite membranes when the content of desorbed dye was also spectrophotometrically monitored and conducted in acidic conditions (HCl aqueous solutions of pH 2). Overall, the introduction of GO in the developed self-standing MALG/AA nanocomposite films exhibited enhanced mechanical properties and increased efficiency for dye removal applications. Their great reutilization potential was highlighted by low drying times and a good ability to release the dye initially adsorbed. Thus, the prepared films could be suitable materials for sustainable and effective water treatment technologies.

Dynamic and Self-Healable Chitosan/Hyaluronic Acid-Based In Situ-Forming Hydrogels.

In situ-forming, biodegradable, and self-healing hydrogels, which maintain their integrity after damage, owing to dynamic interactions, are essential biomaterials for bioapplications, such as tissue engineering and drug delivery. This work aims to develop in situ, biodegradable and self-healable hydrogels based on dynamic covalent bonds between N-succinyl chitosan (S-CHI) and oxidized aldehyde hyaluronic acid (A-HA). A robust effect of the molar ratio of both S-CHI and A-HA was observed on the swelling, mechanical stability, rheological properties and biodegradation kinetics of these hydrogels, being the stoichiometric ratio that which leads to the lowest swelling factor (×12), highest compression modulus (1.1·10−3 MPa), and slowest degradation (9 days). Besides, a rapid (3 s) self-repairing ability was demonstrated in the macro scale as well as by rheology and mechanical tests. Finally, the potential of these biomaterials was evidenced by cytotoxicity essay (>85%).

Electro and magnetoactive printed bi-functional actuators based on alginate hybrid hydrogels.

Soft materials are attracting much attention for the development of biostructures able to mimic the movement of natural systems by remote actuation. Multi-sensitive hydrogels are among the best materials for obtaining dynamic and biocompatible soft structures for soft actuators and related biomedical devices. Nevertheless, bioinks based on naturally occurring and stimuli responsive hydrogels able to be 3D printed continues being a challenge for advanced applications. In this work 3D printable electrically and magnetically responsive, non-cytotoxic, hybrid hydrogels based on alginate and zero monovalent iron nanoparticles (NPs) are presented. The effect of NPs addition on the physico-chemical properties of the hydrogels is addressed, together with its effect on the functional electroactive and magnetoactive response. NPs concentration up to 10 % do not affect the mechanical stability of the gels, while promoting an increase actuation response.

Photocrosslinkable and self-healable hydrogels of chitosan and hyaluronic acid.

Biocompatible and biodegradable hydrogels with biomimetic properties, such as self-repairing, are increasingly interesting for biomedical applications, particularly when they can be printed or in situ formed to mimic extracellular matrix or as personalized implantable devices in tissue regeneration or drug delivery. Photocrosslinkable hydrogels based on methacrylated chitosan (CHIMe) and hyaluronic acid that exhibit according with their composition, tuneable physico-chemical properties are here presented. The study of the conversion, gelation time, mechanical and rheological properties of photopolymerized CHIMe showed an optimal phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) initiator feed (0.1 % w). These photocrosslinkable hydrogels demonstrated being able to promote doubly crosslinked hydrogels with similar Young Moduli regardless the cycles of self-healing processes, and tailored swelling (25-70 swelling factor), mechanical (1 × 10-4-2 × 10-2 MPa) and rheological properties, as a function of polysaccharides relative content. Clear evidences have been found that fast photopolymerization of CHIMe/HA solutions leads to biocompatible (>80 % cell viability), biodegradable (20-24 days in hydrolytic medium) and robust self-healable hydrogels suitable for advanced biomedical and tissue engineering applications.

Self-healing, antibacterial and anti-inflammatory chitosan-PEG hydrogels for ulcerated skin wound healing and drug delivery.

Great efforts have been performed on the production of advanced biomaterials with the combination of self-healing and wound healing properties in implant/tissue engineering biomedical area. Inspired by this idea, chitosan (CHI) based hydrogels can be used to treat a less investigated class of harmful chronic wounds: ulcers or pressure ulcers. Thus, CHI was crosslinked with previously synthesized polyethylene glycol diacid (PEG-diacid) to obtain different CHI-PEG hydrogel formulations with high H-bonding tendency resulting in self-repair ability. Here presented results show biocompatible, antibacterial, anti-inflammatory, and self-healing CHI-PEG hydrogels with a promising future in the treatment of ulcerated wounds by a significant improvement in metabolic activity (94.51 ± 4.38 %), collagen and elastin quantities (2.12 ± 0.63 µg collagen and 4.97 ± 0.61 µg elastin per mg dermal tissue) and histological analysis. Furthermore, cefuroxime (CFX), tetracycline (TCN) and amoxicillin (AMX) antibiotics, and acetylsalicylic acid (ASA) anti-inflammatory agent were sustainedly released for enhancing antibacterial and anti-inflammatory activities of hydrogels.

Drug Delivery from Hyaluronic Acid-BDDE Injectable Hydrogels for Antibacterial and Anti-Inflammatory Applications.

Hyaluronic acid (HA) injectable biomaterials are currently applied in numerous biomedical areas, beyond their use as dermal fillers. However, bacterial infections and painful inflammations are associated with healthcare complications that can appear after injection, restricting their applicability. Fortunately, HA injectable hydrogels can also serve as drug delivery platforms for the controlled release of bioactive agents with a critical role in the control of certain diseases. Accordingly, herein, HA hydrogels were crosslinked with 1 4-butanediol diglycidyl ether (BDDE) loaded with cefuroxime (CFX), tetracycline (TCN), and amoxicillin (AMX) antibiotics and acetylsalicylic acid (ASA) anti-inflammatory agent in order to promote antibacterial and anti-inflammatory responses. The hydrogels were thoroughly characterized and a clear correlation between the crosslinking grade and the hydrogels' physicochemical properties was found after rheology, scanning electron microscopy (SEM), thermogravimetry (TGA), and differential scanning calorimetry (DSC) analyses. The biological safety of the hydrogels, expected due to the lack of BDDE residues observed in 1H-NMR spectroscopy, was also corroborated by an exhaustive biocompatibility test. As expected, the in vitro antibacterial and anti-inflammatory activity of the drug-loaded HA-BDDE hydrogels was confirmed against Staphylococcus aureus by significantly decreasing the pro-inflammatory cytokine levels.

Spontaneous Gelation of Adhesive Catechol Modified Hyaluronic Acid and Chitosan.

Spontaneously formed hydrogels are attracting increasing interest as injectable or wound dressing materials because they do not require additional reactions or toxic crosslinking reagents. Highly valuable properties such as low viscosity before external application, adequate filmogenic capacity, rapid gelation and tissue adhesion are required in order to use them for those therapeutic applications. In addition, biocompatibility and biodegradability are also mandatory. Accordingly, biopolymers, such as hyaluronic acid (HA) and chitosan (CHI), that have shown great potential for wound healing applications are excellent candidates due to their unique physiochemical and biological properties, such as moisturizing and antimicrobial ability, respectively. In this study, both biopolymers were modified by covalent anchoring of catechol groups, and the obtained hydrogels were characterized by studying, in particular, their tissue adhesiveness and film forming capacity for potential skin wound healing applications. Tissue adhesiveness was related to o-quinone formation over time and monitored by visible spectroscopy. Consequently, an opposite effect was observed for both polysaccharides. As gelation advances for HA-CA, it becomes more adhesive, while competitive reactions of quinone in CHI-CA slow down tissue adhesiveness and induce a detriment of the filmogenic properties.

Wound healing and antibacterial chitosan-genipin hydrogels with controlled drug delivery for synergistic anti-inflammatory activity.

Chitosan (CHI) based hydrogels promote wound healing and relieve inflammations and chronic infections. However, in hardly healable ulcers with excessively painful inflammations, anti-inflammatory activity of hydrogels can be enhanced by the sustained release of non-steroidal anti-inflammatory drugs or combining them with antibiotics. Thus, CHI was crosslinked with genipin (GP) to obtain biocompatible hydrogels. Moreover, their antibacterial activity was confirmed against Staphylococcus aureus and Escherichia coli with an almost 100% bacteria reduction and a potential antibacterial efficacy (R > 2). Furthermore, hydrogels effective healing of ulcerated wounds was corroborated by a significant improvement in metabolic activity (95.58 ± 4.40%), collagen and elastin quantities (1.48 ± 0.07 µg collagen and 5.82 ± 0.73 µg elastin per mg dermal tissue) and histological analysis. Finally, the sustained release of acetylsalicylic acid (ASA), cefuroxime (CFX), tetracycline (TCN) and amoxicillin (AMX) were studied, as well as their anti-inflammatory activity. Results confirm the synergistic anti-inflammatory activity by the significant reduction in the amount of pro-inflammatory cytokines when ASA was combined with CFX (5.39 ± 0.81 ng·mL-1 TNF-α), TCN (4.70 ± 0.21 ng·mL-1 TNF-α and 49.06 ± 9.64 ng·mL-1 IL-8), and AMX (2.28 ± 0.36 ng·mL-1 TNF-α, 14.84 ± 5.57 ng·mL-1 IL-8, and total IL-6 removal).

pH-Induced 3D Printable Chitosan Hydrogels for Soft Actuation.

Three-dimensional (3D) printing represents a suitable technology for the development of biomimetic scaffolds for biomedical and tissue engineering applications. However, hydrogel-based inks' printability remains a challenge due to their restricted print accuracy, mechanical properties, swelling or even cytotoxicity. Chitosan is a natural-derived polysaccharide that has arisen as a promising bioink due to its biodegradability, biocompatibility, sustainability and antibacterial properties, among others, as well as its ability to form hydrogels under the influence of a wide variety of mechanisms (thermal, ionic, pH, covalent, etc.). Its poor solubility at physiological pH, which has traditionally restricted its use, represents, on the contrary, the simplest way to induce chitosan gelation. Accordingly, herein a NaOH strong base was employed as gelling media for the direct 3D printing of chitosan structures. The obtained hydrogels were characterized in terms of morphology, chemical interactions, swelling and mechanical and rheological properties in order to evaluate the influence of the gelling solution's ionic strength on the hydrogel characteristics. Further, the influence of printing parameters, such as extrusion speed (300, 600 and 800 mm/min) and pressure (20-35 kPa) and the cytocompatibility were also analyzed. In addition, printed gels show an electro-induced motion due to their polycationic nature, which highlights their potential as soft actuators and active scaffolds.

3D printable self-healing hyaluronic acid/chitosan polycomplex hydrogels with drug release capability.

Multifunctional printable biomaterials are at the base of advanced biomedical applications. Chitosan (CHI) and hyaluronic acid (HA) allow the development of polycomplex hydrogels with tailorable properties, including self-healing and controlled drug release. This work correlates and optimizes the mucoadhesive, swelling, biodegradation, mechanical and rheological properties of HA/CHI polycomplex hydrogels with synthesis parameters such as polysaccharide content and complexation time, according to the interaction forces established between both polyelectrolytes. Related to these dynamic forces, the self-healing ability of the hydrogels was investigated together with the potential of the HA/CHI polycomplex hydrogels for 3D printing. Finally, their capability to modulate and promote controlled release of a variety of drugs (anionic and anti-inflammatory sodium diclofenac and the neutral antibiotic rifampicin) was demonstrated. Thus, the reported tunable properties, self-repair ability, printability and drug release properties, demonstrate the suitability of HA/CHI hydrogels for advanced biomedical applications.

Biocompatible hyaluronic acid-divinyl sulfone injectable hydrogels for sustained drug release with enhanced antibacterial properties against Staphylococcus aureus.

Hyaluronic acid (HA) solutions were crosslinked with divinyl sulfone (DVS) and subsequently loaded with antibiotic molecules to obtain biocompatible and antibacterial injectable hydrogels. The crosslinking degree of the hydrogels was modulated by varying the reaction time and the HA:DVS weight ratio. Synthesized HA-DVS hydrogels were characterized by their rheological properties, pore size, swelling capacity and hydrolytic and thermal degradation. Biocompatibility was assessed by measuring pH, osmolality and by in vitro cytotoxic assay. Acetyl salicylic (AAS) loaded hydrogels display anti-inflammatory properties in vitro, whereas cefuroxime (CFX), tetracycline (TCN) and amoxicillin (AMX) loaded hydrogels show in vitro antibacterial activity against Staphylococcus aureus. The combine use of antibiotics and AAS produces a synergic effect that reduces the S. aureus population up to a log10 reduction (R) of 5.55. Overall results show that antibiotic/AAS loaded HA-DVS hydrogels could be effectively used to combat S. aureus infections and to increase the antibacterial activity of antibiotics commonly used against S. aureus.

Antibacterial catechol-based hyaluronic acid, chitosan and poly (N-vinyl pyrrolidone) coatings onto Ti6Al4V surfaces for application as biomedical implant.

Bacterial contamination in implanted biomedical devices is a critical daily concern. The most used material for permanent implant in biomedical field is Ti6Al4V alloy due to its beneficial mechanical properties and high biocompatibility. Accordingly, in this work different polymeric antibacterial coatings poly(N-vinyl pyrrolidone) (PVP), hyaluronic acid (HA) and chitosan (CHI) were developed and comparatively analysed for Ti6Al4V surface covering. The adhesion of these coatings to Ti6Al4V substrates were carried out after the conjugation of these polymers with the so well-known bioadhesive properties of catechol (CA) anchor group. These surface modifications were characterized by X-ray photoelectronic spectroscopy, contact angle measurements and atomic force microscopy. In addition, the stability of CA-conjugated polymeric coatings was compared with the coatings formed with unconjugated polymers. Finally, the cytocompatibility and antibacterial properties against gram-positive and gram-negative strains on coated Ti6Al4V substrates were assessed confirming the effectiveness of these polymeric coatings against bacterial infections for future applications in protecting biomedical implants.

Tough Hydrogels Based on Maleic Anhydride, Bulk Properties Study and Microfiber Formation by Electrospinning.

Hydrogels present a great number of advantages, such as their swelling capacity or their capability to mimic tissues, which make them very interesting biomaterials. However, one of their main disadvantages is their lack of good mechanical properties, which could limit some of their applications. Several strategies have been carried out to develop hydrogels with enhanced mechanical properties, but many of the suggested synthetic pathways to improve this property are expensive and time consuming. In this work, we studied an easy synthetic path to produce tough hydrogels based on different maleic anhydride copolymers crosslinked with polyethylenglycol. The effect of the comonomers in the mechanical properties has been studied, their excellent mechanical properties, good swelling behavior and thermal stability being remarkable. In addition, in order to evaluate their possible applications as scaffolds or in wound healing applications, microsized fibers have been fabricated by electrospinning.

Green alternative cosolvents to N-methyl-2-pyrrolidone in water polyurethane dispersions.

N-Methyl-2-pyrrolidone is a toxic dipolar aprotic solvent widely used in the synthesis of polyurethane dispersions (PUD). Since legislation strongly restricts this substance, green alternatives are essential. Dihydrolevoglucosenone and gamma valerolactone demonstrate comparable performance to that of NMP as cosolvent in the synthesis and the film forming process of PUD.

Bioactive Coatings on Titanium: A Review on Hydroxylation, Self-Assembled Monolayers (SAMs) and Surface Modification Strategies.

Titanium (Ti) and its alloys have been demonstrated over the last decades to play an important role as inert materials in the field of orthopedic and dental implants. Nevertheless, with the widespread use of Ti, implant-associated rejection issues have arisen. To overcome these problems, antibacterial properties, fast and adequate osseointegration and long-term stability are essential features. Indeed, surface modification is currently presented as a versatile strategy for developing Ti coatings with all these challenging requirements and achieve a successful performance of the implant. Numerous approaches have been investigated to obtain stable and well-organized Ti coatings that promote the tailoring of surface chemical functionalization regardless of the geometry and shape of the implant. However, among all the approaches available in the literature to functionalize the Ti surface, a promising strategy is the combination of surface pre-activation treatments typically followed by the development of intermediate anchoring layers (self-assembled monolayers, SAMs) that serve as the supporting linkage of a final active layer. Therefore, this paper aims to review the latest approaches in the biomedical area to obtain bioactive coatings onto Ti surfaces with a special focus on (i) the most employed methods for Ti surface hydroxylation, (ii) SAMs-mediated active coatings development, and (iii) the latest advances in active agent immobilization and polymeric coatings for controlled release on Ti surfaces.