Biopolymers for Tissue Engineering: Crosslinking, Printing Techniques, and Applications.

Currently, tissue engineering has been dedicated to the development of 3D structures through bioprinting techniques that aim to obtain personalized, dynamic, and complex hydrogel 3D structures. Among the different materials used for the fabrication of such structures, proteins and polysaccharides are the main biological compounds (biopolymers) selected for the bioink formulation. These biomaterials obtained from natural sources are commonly compatible with tissues and cells (biocompatibility), friendly with biological digestion processes (biodegradability), and provide specific macromolecular structural and mechanical properties (biomimicry). However, the rheological behaviors of these natural-based bioinks constitute the main challenge of the cell-laden printing process (bioprinting). For this reason, bioprinting usually requires chemical modifications and/or inter-macromolecular crosslinking. In this sense, a comprehensive analysis describing these biopolymers (natural proteins and polysaccharides)-based bioinks, their modifications, and their stimuli-responsive nature is performed. This manuscript is organized into three sections: (1) tissue engineering application, (2) crosslinking, and (3) bioprinting techniques, analyzing the current challenges and strengths of biopolymers in bioprinting. In conclusion, all hydrogels try to resemble extracellular matrix properties for bioprinted structures while maintaining good printability and stability during the printing process.

Chemical Composition of Macroalgae Polysaccharides from Galician and Portugal Coasts: Seasonal Variations and Biological Properties.

Crude polysaccharides extracted from the Codium sp. and Osmundea sp. macroalgae collected in different seasons (winter, spring and summer) from the Galician and North Portugal coasts were characterised, aiming to support their biomedical application to wound healing. An increase in polysaccharides' sulphate content was registered from winter to summer, and higher values were obtained for Osmundea sp. In turn, the monosaccharide composition constantly changed with a decrease in glucose in Osmundea sp. from spring to winter. For Codium sp., a higher increase was noticed regarding glucose content in the Galician and Portugal coasts. Galactose was the major monosaccharide in all the samples, remaining stable in all seasons and collection sites. These results corroborate the sulphate content and antioxidant activity, since the Osmundea sp.-derived polysaccharides collected in summer exhibited higher scavenging radical ability. The biocompatibility and wound scratch assays revealed that the Osmundea sp. polysaccharide extracted from the Portugal coast in summer possessed more potential for promoting fibroblast migration. This study on seasonal variations of polysaccharides, sulphate content, monosaccharide composition and, consequently, biological properties provides practical guidance for determining the optimal season for algae harvest to standardise preparations of polysaccharides for the biomedical field.

Interaction of Near-Infrared (NIR)-Light Responsive Probes with Lipid Membranes: A Combined Simulation and Experimental Study.

Cancer is considered a major societal challenge for the next decade worldwide. Developing strategies for simultaneous diagnosis and treatment has been considered a promising tool for fighting cancer. For this, the development of nanomaterials incorporating prototypic near-infrared (NIR)-light responsive probes, such as heptamethine cyanines, has been showing very promising results. The heptamethine cyanine-incorporating nanomaterials can be used for a tumor's visualization and, upon interaction with NIR light, can also produce a photothermal/photodynamic effect with a high spatio-temporal resolution and minimal side effects, leading to an improved therapeutic outcome. In this work, we studied the interaction of 12 NIR-light responsive probes with lipid membrane models by molecular dynamics simulations. We performed a detailed characterization of the location, orientation, and local perturbation effects of these molecules on the lipid bilayer. Based on this information, the probes were divided into two groups, predicting a lower and higher perturbation of the lipid bilayer. From each group, one molecule was selected for testing in a membrane leakage assay. The experimental data validate the hypothesis that molecules with charged substituents, which function as two polar anchors for the aqueous phase while spanning the membrane thickness, are more likely to disturb the membrane by the formation of defects and pores, increasing the membrane leakage. The obtained results are expected to contribute to the selection of the most suitable molecules for the desired application or eventually guiding the design of probe modifications for achieving an optimal interaction with tumor cell membranes.

Three-Dimensionally Printed Hydrogel Cardiac Patch for Infarct Regeneration Based on Natural Polysaccharides.

Myocardial infarction is one of the more common cardiovascular diseases, and remains the leading cause of death, globally. Hydrogels (namely, those using natural polymers) provide a reliable tool for regenerative medicine and have become a promising option for cardiac tissue regeneration due to their hydrophilic character and their structural similarity to the extracellular matrix. Herein, a functional ink based on the natural polysaccharides Gellan gum and Konjac glucomannan has, for the first time, been applied in the production of a 3D printed hydrogel with therapeutic potential, with the goal of being locally implanted in the infarcted area of the heart. Overall, results revealed the excellent printability of the bioink for the development of a stable, porous, biocompatible, and bioactive 3D hydrogel, combining the specific advantages of Gellan gum and Konjac glucomannan with proper mechanical properties, which supports the simplification of the implantation process. In addition, the structure have positive effects on endothelial cells' proliferation and migration that can promote the repair of injured cardiac tissue. The results presented will pave the way for simple, low-cost, and efficient cardiac tissue regeneration using a 3D printed hydrogel cardiac patch with potential for clinical application for myocardial infarction treatment in the near future.

Animal-derived products in science and current alternatives.

More than fifty years after the 3Rs definition and despite the continuous implementation of regulatory measures, animals continue to be widely used in basic research. Their use comprises not only in vivo experiments with animal models, but also the production of a variety of supplements and products of animal origin for cell and tissue culture, cell-based assays, and therapeutics. The animal-derived products most used in basic research are fetal bovine serum (FBS), extracellular matrix proteins such as Matrigel™, and antibodies. However, their production raises several ethical issues regarding animal welfare. Additionally, their biological origin is associated with a high risk of contamination, resulting, frequently, in poor scientific data for clinical translation. These issues support the search for new animal-free products able to replace FBS, Matrigel™, and antibodies in basic research. In addition, in silico methodologies play an important role in the reduction of animal use in research by refining the data previously to in vitro and in vivo experiments. In this review, we depicted the current available animal-free alternatives in in vitro research.

Silk Sericin: A Promising Sustainable Biomaterial for Biomedical and Pharmaceutical Applications.

Silk is a natural composite fiber composed mainly of hydrophobic fibroin and hydrophilic sericin, produced by the silkworm Bombyx mori. In the textile industry, the cocoons of B. mori are processed into silk fabric, where the sericin is substantially removed and usually discarded in wastewater. This wastewater pollutes the environment and water sources. However, sericin has been recognized as a potential biomaterial due to its biocompatibility, immunocompatibility, biodegradability, anti-inflammatory, antibacterial, antioxidant and photoprotective properties. Moreover, sericin can produce hydrogels, films, sponges, foams, dressings, particles, fibers, etc., for various biomedical and pharmaceutical applications (e.g., tissue engineering, wound healing, drug delivery, cosmetics). Given the severe environmental pollution caused by the disposal of sericin and its beneficial properties, there has been growing interest in upcycling this biomaterial, which could have a strong and positive economic, social and environmental impact.

Ciprofloxacin-Loaded Zein/Hyaluronic Acid Nanoparticles for Ocular Mucosa Delivery.

Bacterial conjunctivitis is a worldwide problem that, if untreated, can lead to severe complications, such as visual impairment and blindness. Topical administration of ciprofloxacin is one of the most common treatments for this infection; however, topical therapeutic delivery to the eye is quite challenging. To tackle this, nanomedicine presents several advantages compared to conventional ophthalmic dosage forms. Herein, the flash nanoprecipitation technique was applied to produce zein and hyaluronic acid nanoparticles loaded with ciprofloxacin (ZeinCPX_HA NPs). ZeinCPX_HA NPs exhibited a hydrodynamic diameter of <200 nm and polydispersity index of <0.3, suitable for ocular drug delivery. In addition, the freeze-drying of the nanoparticles was achieved by using mannitol as a cryoprotectant, allowing their resuspension in water without modifying the physicochemical properties. Moreover, the biocompatibility of nanoparticles was confirmed by in vitro assays. Furthermore, a high encapsulation efficiency was achieved, and a release profile with an initial burst was followed by a prolonged release of ciprofloxacin up to 24 h. Overall, the obtained results suggest ZeinCPX_HA NPs as an alternative to the common topical dosage forms available on the market to treat conjunctivitis.

Single-Step Self-Assembly of Zein-Honey-Chitosan Nanoparticles for Hydrophilic Drug Incorporation by Flash Nanoprecipitation.

Zein- and chitosan-based nanoparticles have been described as promising carrier systems for food, biomedical and pharmaceutical applications. However, the manufacture of size-controlled zein and chitosan particles is challenging. In this study, an adapted anti-solvent nanoprecipitation method was developed. The effects of the concentration of zein and chitosan and the pH of the collection solution on the properties of the zein-honey-chitosan nanoparticles were investigated. Flash nanoprecipitation was demonstrated as a rapid, scalable, single-step method to achieve the self-assembly of zein-honey-chitosan nanoparticles. The nanoparticles size was tuned by varying certain formulation parameters, including the total concentration and ratio of the polymers. The zein-honey-chitosan nanoparticles' hydrodynamic diameter was below 200 nm and the particles were stable for 30 days. Vitamin C was used as a hydrophilic model substance and efficiently encapsulated into these nanoparticles. This study opens a promising pathway for one-step producing zein-honey-chitosan nanoparticles by flash nanoprecipitation for hydrophilic compounds' encapsulation.

Osmundea sp. macroalgal polysaccharide-based nanoparticles produced by flash nanocomplexation technique.

The macroalgae-derived polysaccharides' biological potential has been explored due to their attractive intrinsic properties such as biocompatibility, biodegradability, and their ability to conjugate with other compounds. In particular, in the drug delivery systems field, the anionic macroalgae polysaccharides have been combined with cationic compounds through ionotropic gelation and/or bulk mixing. However, these techniques did not assure reproducibility, and the stability of nanoparticles is undesired. To overcome these limitations, herein, the polysaccharide extracted from Osmundea sp. was used to produce nanoparticles through the flash nanocomplexation technique. This approach rapidly mixed the negative charge of macroalgae polysaccharide with a positive chitosan charge on a millisecond timescale. Further, diclofenac (an anti-inflammatory drug) was also incorporated into complex nanoparticles. Overall, the gathered data showed that hydrodynamic diameter nanoparticles values lower than 100 nm, presenting a narrow size distribution and stability. Also, the diclofenac exhibited a targeted and sustained release profile in simulating inflammatory conditions. Likewise, the nanoparticles showed excellent biological properties, evidencing their suitability to be used to treat inflammatory skin diseases.

Oromucosal Alginate Films with Zein Nanoparticles as a Novel Delivery System for Digoxin.

Digoxin is a hydrophobic drug used for the treatment of heart failure that possesses a narrow therapeutic index, which raises safety concerns for toxicity. This is of utmost relevance in specific populations, such as the elderly. This study aimed to demonstrate the potential of the sodium alginate films as buccal drug delivery system containing zein nanoparticles incorporated with digoxin to reduce the number of doses, facilitating the administration with a quick onset of action. The film was prepared using the solvent casting method, whereas nanoparticles by the nanoprecipitation method. The nanoparticles incorporated with digoxin (0.25 mg/mL) exhibited a mean size of 87.20 ± 0.88 nm, a polydispersity index of 0.23 ± 0.00, and a zeta potential of 21.23 ± 0.07 mV. Digoxin was successfully encapsulated into zein nanoparticles with an encapsulation efficiency of 91% (±0.00). Films with/without glycerol and with different concentrations of ethanol were produced. The sodium alginate (SA) films with 10% ethanol demonstrated good performance for swelling (maximum of 1474%) and mechanical properties, with a mean tensile strength of 0.40 ± 0.04 MPa and an elongation at break of 27.85% (±0.58), compatible with drug delivery application into the buccal mucosa. The current study suggests that SA films with digoxin-loaded zein nanoparticles can be an effective alternative to the dosage forms available on the market for digoxin administration.

Photocurable Polymeric Blends for Surgical Application.

The preparation of photocrosslinkable bioadhesives synthesized from oligomers of lactic acid and polycaprolactone (PCL), both functionalized with 2-isocyanoethyl acrylate (AOI), were studied. The obtained modified macromers of LA-AOI (mLA) and PCL-AOI (mCL) were chemically characterized by 1H NMR and used to formulate polymeric blends with different mass proportions, 1:1, 1:2 and 2:1, respectively. Subsequently, the produced blends were crosslinked, considering two UV irradiation times: 30 and 120 s. After their production, the thermal and mechanical properties of bioadhesives were assessed, where upon the rheology, gel content, hydrolytic degradation and dynamic contact angles were determined. Furthermore, the cytotoxic profile of bioadhesives was evaluated in contact with human dermal fibroblasts cells, whereas their antibacterial effect was studied monitoring Escherichia coli and S. aureus growth. Overall, flexible and resistant films were obtained, presenting promising features to be used as surgical bioadhesives.

Injectable in situ forming thermo-responsive graphene based hydrogels for cancer chemo-photothermal therapy and NIR light-enhanced antibacterial applications.

Functionalized graphene oxide (GO) and reduced GO (rGO) based nanomaterials hold a great potential for cancer photothermal therapy. However, their systemic administration has been associated with an accelerated blood clearance and/or with suboptimal tumor uptake. To address these limitations, the local delivery of GO/rGO to the tumor site by 3D matrices arises as a promising strategy. In this work, injectable chitosan-agarose in situ forming thermo-responsive hydrogels incorporating GO (thermogel-GO) or rGO (thermogel-rGO) were prepared for the first time. The hydrogels displayed suitable injectability and gelation time, as well as good physicochemical properties and cytocompatibility. When irradiated with near infrared (NIR) light, the thermogel-rGO produced a 3.8-times higher temperature increase than thermogel-GO, thus decreasing breast cancer cells' viability to 60%. By incorporating an optimized molar ratio of the Doxorubicin:Ibuprofen combination on thermogel-rGO, this formulation mediated a chemo-photothermal effect that further diminished cancer cells' viability to 34%. In addition, the hydrogels' antibacterial activity was further enhanced upon NIR laser irradiation, which is an important feature considering the possible risk of infection at the site of administration. Overall, thermogel-rGO is a promising injectable in situ forming hydrogel for combinatorial chemo-photothermal therapy of breast cancer cells and NIR light enhanced antibacterial applications.

Hyaluronic acid-Based wound dressings: A review.

Hyaluronic acid (HA), a non-sulfated glycosaminoglycan (GAG), is a major component of skin extracellular matrix (ECM) and it is involved in the inflammatory response, angiogenesis, and tissue regeneration process. Due to the intrinsic properties of HA (such as biocompatibility, biodegradability and hydrophilic character), it has been used to produce different wound dressings, namely sponges, films, hydrogels, and electrospun membranes. Herein, an overview of the different HA-based wound dressings that have been produced so far is provided as well as the future directions regarding the strategies aimed to improve the mechanical stability of HA-based wound dressings, along with the incorporation of biomolecules intended to ameliorate their biological performance during the healing process.

Hyaluronic acid and vitamin E polyethylene glycol succinate functionalized gold-core silica shell nanorods for cancer targeted photothermal therapy.

Gold-core mesoporous silica shell (AuMSS) nanorods unique physicochemical properties makes them versatile and promising nanomedicines for cancer photothermal therapy. Nevertheless, these nanomaterials present a reduced half-life in the blood and poor specificity towards the tumor tissue. Herein, d-α-Tocopherol polyethylene glycol 1000 succinate (TPGS) and Hyaluronic Acid (HA) were combined for the first time to improve the AuMSS nanorods biological performance. The obtained results revealed that AuMSS surface functionalization induced the surface charge neutralization, from -28 ±â€¯10 mV to -3 ±â€¯5 mV and -10 ±â€¯4 mV for AuMSS-TPGS-HA (1:1) and (4:1) formulations, without impacting on nanomaterials' photothermal capacity. Moreover, the AuMSS functionalization improved the nanomaterials hemocompatibility and selectivity towards the cancer cells, particularly in the AuMSS-TPGS-HA (4:1) formulation. Furthermore, both formulations were able to mediate an on-demand photothermal effect, that induced the HeLa cancer cells death, confirming its potential for being applied as targeted multifunctional theragnostic nanomedicines.

Xanthan Gum-Konjac Glucomannan Blend Hydrogel for Wound Healing.

Hydrogels are considered to be the most ideal materials for the production of wound dressings since they display a three-dimensional structure that mimics the native extracellular matrix of skin as well as a high-water content, which confers a moist environment at the wound site. Until now, different polymers have been used, alone or blended, for the production of hydrogels aimed for this biomedical application. From the best of our knowledge, the application of a xanthan gum-konjac glucomannan blend has not been used for the production of wound dressings. Herein, a thermo-reversible hydrogel composed of xanthan gum-konjac glucomannan (at different concentrations (1% and 2% w/v) and ratios (50/50 and 60/40)) was produced and characterized. The obtained data emphasize the excellent physicochemical and biological properties of the produced hydrogels, which are suitable for their future application as wound dressings.

Poly (vinyl alcohol)/chitosan layer-by-layer microneedles for cancer chemo-photothermal therapy.

The combination of photothermal and chemo- therapies displays a high potential to increase the efficacy of the cancer treatments or even promote their eradication. In this study, the micromoulding and electrospraying techniques were combined to produce polyvinylpyrrolidone microneedles coated with chitosan and poly (vinyl alcohol) for mediating the delivery of doxorubicin and AuMSS nanorods (Dox@MicroN) to cancer cells. The microneedles' physicochemical characterization demonstrated that the electrospraying technique can be used to produce a layer-by-layer coating consisting of layers of doxorubicin-loaded chitosan and AuMSS enriched poly (vinyl alcohol). Further, the Dox@MicroN patches presented a good photothermal capacity leading to a temperature increase of 12 °C under near-infrared irradiation (808 nm, 1.7 W/cm-2 for 5 min), which in conjugation with the chitosan' pH sensitivity could be used to control the doxorubicin release. Moreover, the microneedles were able to penetrate the tumor-mimicking agarose gel and promote a layer dependent drug release. Additionally, the Dox@MicroN patches' capacity to simultaneously mediate the chemo- and photothermal-therapies rendered a superior cytotoxic effect against the cervical cancer cells. Overall, the Dox@MicroN patches demonstrated to be a simple macroscale delivery device that can be used to mediate the local administration of new drug-photothermal combinations, avoiding all the issues related to the systemic administration of anti-cancer therapeutics.

Microneedle-based delivery devices for cancer therapy: A review.

Macroscale delivery systems that can be locally implanted on the tumor tissue as well as avoid all the complications associated to the systemic delivery of therapeutics have captured researchers' attention, in recent years. Particularly, the microneedle-based devices can be used to efficiently deliver both small and macro-molecules, like chemotherapeutics, proteins, and genetic material, along with nanoparticle-based anticancer therapies. Such capacity prompted the application of microneedle devices for the development of new anticancer vaccines that can permeate the tumor tissue and simultaneously improve the effectiveness of therapeutic agents. Based on the promising results demonstrated by the microneedle systems in the local administration of anticancer therapeutics, this review summarizes the different microneedle formulations developed up to now aimed for application on cancer therapy (mphasizing those produced with polymers). Additionally, the microneedles' general properties, type of therapeutic approach and its main advantages are also highlighted.

Development of a poly(vinyl alcohol)/lysine electrospun membrane-based drug delivery system for improved skin regeneration.

Nanofiber-based wound dressings are currently being explored as delivery systems of different biomolecules for avoiding skin infections as well as improve/accelerate the healing process. In the present work, a nanofibrous membrane composed of poly(vinyl alcohol) (PVA) and lysine (Lys) was produced by using the electrospinning technique. Further, anti-inflammatory (ibuprofen (IBP)) and antibacterial (lavender oil (LO)) agents were incorporated within the electrospun membrane through blend electrospinning and surface physical adsorption methods, respectively. The obtained results demonstrated that the PVA_Lys electrospun membranes incorporating IBP or LO displayed the suitable morphological, mechanical and biological properties for enhancing the wound healing process. Moreover, the controlled and sustained release profile attained for IBP was appropriate for the duration of the wound healing inflammatory phase, whereas the initial burst release of LO is crucial to prevent wound bacterial contamination. Indeed, the PVA_Lys_LO electrospun membranes were able to mediate a strong antibacterial activity against both S. aureus and P. aeruginosa, without compromising human fibroblasts viability. Overall, the gathered data emphasizes the potential of the PVA_Lys electrospun membranes-based drug delivery systems to be used as wound dressings.

Production and characterization of a novel asymmetric 3D printed construct aimed for skin tissue regeneration.

Skin is a complex organ that act as the first protective barrier against any external threat. After an injury occurs, its structure and functions must be re-established as soon as possible. Among different available skin substitutes (epidermal, dermal and dermo-epidermal), none of them is fully capable of reproducing/re-establishing all the features and functions of native skin. Herein, a three-dimensional skin asymmetric construct (3D_SAC) was produced using electrospinning and 3D bioprinting techniques. A poly(caprolactone) and silk sericin blend was electrospun to produce a top layer aimed to mimic the epidermis features, i.e. to provide a protective barrier against dehydration and hazard agents. In turn, the dermis like layer was formed by printing layer-by-layer a chitosan/sodium alginate hydrogel. The results obtained in the in vitro assays revealed that the 3D_SAC display a morphology, porosity, mechanical properties, wettability, antimicrobial activity and a cytotoxic profile that grants their application as a skin substitute during the healing process.

An overview of electrospun membranes loaded with bioactive molecules for improving the wound healing process.

Nowadays, despite the intensive research performed in the area of skin tissue engineering, the treatment of skin lesions remains a big challenge for healthcare professionals. In fact, none of the wound dressings currently used in the clinic is capable of re-establishing all the native features of skin. An ideal wound dressing must confer protection to the wound from external microorganisms, chemical, and physical aggressions, as well as promote the healing process by stimulating the cell adhesion, differentiation, and proliferation. In recent years different types of wound dressings (such as films, hydrocolloids, hydrogels, micro/nano fibers) have been developed. Among them, electrospun nanofibrous membranes due to their intrinsic properties like high surface area-to-volume ratio, porosity and structural similarity with the skin extracellular matrix have been regarded as highly promising for wound dressings applications. Additionally, the nanofibers available in these membranes can act as drug delivery systems, which prompted the incorporation of biomolecules within their structure to prevent skin infections as well as improve the healing process. In this review, examples of different bioactive molecules that have been loaded on polymeric nanofibers are presented, highlighting the antibacterial biomolecules (e.g. antibiotics, silver nanoparticles and natural extracts-derived products) and the molecules capable of enhancing the healing process (e.g. growth factors, vitamins, and anti-inflammatory molecules).