There are many restrictions on topical medications for the oral cavity. Various factors affect the topical application of drugs in the oral cavity, an open and complex environment. The complex physical and chemical environment of the oral cavity, such as saliva and food, will influence the effect of free drugs. Therefore, drug delivery systems have served as supporting structures or as carriers loading active ingredients, such as antimicrobial agents and growth factors (GFs), to promote antibacterial properties, tissue regeneration, and engineering for drug diffusion. These drug delivery systems are considered in the prevention and treatment of dental caries, periodontal disease, periapical disease, the delivery of anesthetic drugs, etc. These carrier materials are designed in different ways for clinical application, including nanoparticles, hydrogels, nanofibers, films, and scaffolds. This review aimed to summarize the advantages and disadvantages of different carrier materials. We discuss synthesis methods and their application scope to provide new perspectives for the development and preparation of more favorable and effective local oral drug delivery systems.

One of the greatest challenges associated with tissue engineering is the development of biomimetic three-dimensional structures that closely emulate the complexity and functionality of native tissues. The main objective of this work was the biofabrication of biomimetic three-dimensional structures for tissue engineering, aiming to achieve enhanced emulation of native tissue complexity and functionality, by the addition of different fractions of carbon dots. In an initial phase this work consists of the state of the art related to tissue engineering, biomaterials and scaffolds. Research was also carried out on biofabrication and its main steps and the extrusion-based bioprinting process was described. In another phase, the experimental plan was carried out, containing the materials and methods used to achieve the obtained results, such as compressive tests and 3D printing methods. Compression tests were carried out on 15 types of hydrogels composed of ColMA, CDs, and GQDs and the influence of their addition to the hydrogels was studied. It was found that the viscosity of the prepared solutions increased linearly with increasing ColMA concentration. For the same ColMA concentration, the hydrogels composed of ColMA and CDs, as the CDs concentration increased, the Young’s modulus increased. The addition of GQDs did not benefit all hydrogels, as is the case with hydrogels with a concentration of 1% ColMA, causing a decrease in Young’s modulus. 3D printing depends on several parameters such as ink viscosity, feed rate, printing pressure, needle diameter. If the feed rate is too slow, an excessive amount of material is pushed out, leading to the possibility of printing undefined structures that lack microarchitecture. On the other hand, if the feed rate is too high, there may not be enough material extruded from the nozzle, resulting in the inability to obtain any printed structures. The results obtained prove that the addition of 5% CDs can be beneficial for 3D printing, obtaining filaments with excellent uniformity, well-defined lines and angles, and good filament width.

Over the past decade, 3D printing has gained increasing popularity, being a technique capable of producing well-defined tissue-like structures. One of its most appealing features is the ability to create personalized therapies to meet the specific demands of individual patients. However, challenges including the selection of materials, as well as crosslinking strategies still need to be addressed, in order to enhance the inks characteristics and simultaneously create printable, robust, and cytocompatible biomaterials. Hence, this study reports the development of natural-based photoclickable inks, composed of hyaluronic acid (HA) and proteins [bovine serum albumin (BSA) and human platelet lysates (hPL)] for 3D extrusion printing, taking advantage of the versatility, efficiency, and fast kinetics of thiol-ene click chemistry. To this end, HA was modified with norbornene (HA-Nor) moieties, while proteins were thiolated (BSA-SH and hPL-SH). 1H-NMR and ATR-FTIR analysis of the resulting polymers, HA-Nor, BSA-SH and hPL-SH, confirmed the success of their modification. These polymers were mixed, varying the protein concentration and, subsequently, bio-orthogonally photo-crosslinked, using visible light and LAP as photoiniator, creating hydrogel cell-friendly networks. Rheological characterization affirmed the chemistry’s potential in rapidly gelling networks (t < 20 s) achieving an elastic modulus, G’, of approximately 104 Pa, highlighting its suitability for 3D printing applications. Moreover, the mechanical characterization of the hydrogels revealed that those containing higher BSA-SH concentration were more robust, indicating the presence of more crosslinking points, as expected. The mechanical and rheological properties of the inks enabled 3D extrusion printing. Additionally, direct and indirect contact of hASCs with the materials through seeding, confirmed their cytocompatibility and non-toxicity, maintaining cell viability up to 14 days, which was assessed through fluorescence microscopy images and metabolic activity. Therefore, HA-Nor/BSA-SH and HA-Nor/hPL-SH inks are expected to be promising future bioinks for regenerative medicine and tissue engineering of soft tissues, opening avenues for personalized medicine.

Three-dimensional (3D) cell culture platforms have emerged as potential alternatives to the two-dimensional platforms traditionally used, due to their ability to recapitulate the native microenvironment more accurately. Inspired by the extracellular matrix (ECM) of tissues researchers are increasingly focus on developing models that mimic this structure. Hydrogels are hydrophilic polymeric networks capable of retaining large amounts of water, allowing nutrient and oxygen exchange, as well as cell adhesion and proliferation. Currently, Matrigel® is the most widely used commercial biomaterial for 3D cell culture, but due to its animal origin it is not efficient at accurately recapitulating the human microenvironment, resulting in a reduction in the clinical value of the in vitro results. In 2018, Metatissue® saw this gap as an opportunity to integrate into the market cell culture platforms derived from human blood and perinatal tissues. Hydrogels derived from amniotic membrane (AM), capable of effectively recapitulate the microenvironment of the human body, are being optimized for future commercialization. However, the production yields of the product per AM are very low.

The well-known synergetic interplay between the skeletal and immune systems has changed the design of advanced bone tissue engineering strategies. The immune system is essential during the bone lifetime, with macrophages playing multiple roles in bone healing and biomaterial integration. If in the past, the most valuable aspect of implants was to avoid immune responses of the host, nowadays, it is well-established how important are the crosstalks between immune cells and bone-engineered niches for an efficient regenerative process to occur. For that, it is essential to recapitulate the multiphenotypic cellular environment of bone tissue when designing new approaches. Indeed, the lack of osteoimmunomodulatory knowledge may be the explanation for the poor translation of biomaterials into clinical practice. Thus, smarter hydrogels incorporating immunomodulatory bioactive factors, stem cells, and immune cells are being proposed to develop a new generation of bone tissue engineering strategies. This review highlights the power of immune cells to upgrade the development of innovative engineered strategies, mainly focusing on orthopaedic and dental applications.

Osteoarthritis (OA) is a frequent degenerative condition of the articular cartilage characterized by hypertrophic changes in the bone. It is the most typical joint disorder, affecting approximately 300 million worldwide. Due to this and the lack of cartilage’s healing capabilities, many studies have been conducted to find a cure for OA. Several clinical procedures with the aim to reduce the pain or lack of movement have already been applied in patients with OA, and even though the results seem promising in the early post-operatory, they are never permanent. Novel techniques for cartilage regeneration have been studied, including using natural polymer-based hydrogels, since natural polymers have many different and unique properties advantageous for cartilage regeneration. Chitosan (CHI) is the most promising biopolymer for the treatment of OA since it is biocompatible, biodegradable, and composed of D-glucosamine units. This monomer is a natural building block of the cartilage and plays a massive role in its regeneration, being responsible for attenuating inflammation, inhibiting the activity of different enzymes responsible for cartilage degradation and repairing damaged tissues. Therefore, the work developed in this master's thesis aims to prepare a CHI hydrogel that promotes cartilage regeneration, by attenuating inflammation, from the phased release of D-glucosamine units by the action of encapsulated enzymes. Methacrylated low molecular weight CHI (LmwMACHI) with 22% modification degree was obtained by modification of CHI with methacrylic anhydride. LmwMACHI can form hydrogels through covalent bonds between its methacrylic groups when exposed to UV irradiation. Mechanical characterization evidenced that Double-Network (DN) hydrogels presented better results than LmwMACHI hydrogels, in Young’s modulus and toughness, and the same happened with the 2% MACHI DNWTPP 50/50 hydrogels. Exo-β-D-glucosaminidase and α-amylase were chosen, out of a group of tested enzymes, to be encapsulated in a 2% MACHI DNWTPP 50/50 hydrogel and demonstrated a phased release of D-glucosamine monomers. To evaluate the anti-inflammatory effect, biological assays of in vitro cell culture were performed using the hydrogel 2% MACHI DNWTPP 50/50 with the encapsulated enzymes. The results indicated that the amount of D-glucosamine released by the hydrogels was not effective in preventing inflammation. To conclude, the developed hydrogels are promising, but lack optimization in the release of D-glucosamine.

Three-dimensional (3D) bioprinting is an innovative technology in the biomedical field, allowing the fabrication of living constructs through an approach of layer-by-layer deposition of cell-laden inks, the so-called bioinks. An ideal bioink should possess proper mechanical, rheological, chemical, and biological characteristics to ensure high cell viability and the production of tissue constructs with dimensional stability and shape fidelity. Among the several types of bioinks, hydrogels are extremely appealing as they have many similarities with the extracellular matrix, providing a highly hydrated environment for cell proliferation and tunability in terms of mechanical and rheological properties. Hydrogels derived from natural polymers, and polysaccharides, in particular, are an excellent platform to mimic the extracellular matrix, given their low cytotoxicity, high hydrophilicity, and diversity of structures. In fact, polysaccharide-based hydrogels are trendy materials for 3D bioprinting since they are abundant and combine adequate physicochemical and biomimetic features for the development of novel bioinks. Thus, this review portrays the most relevant advances in polysaccharide-based hydrogel bioinks for 3D bioprinting, focusing on the last five years, with emphasis on their properties, advantages, and limitations, considering polysaccharide families classified according to their source, namely from seaweed, higher plants, microbial, and animal (particularly crustaceans) origin.

Spinal cord injury (SCI) is an extremely serious condition which leads to a drastic decrease on patient mobility. Neural tissue engineering (NTE) has been trying to promote solutions by combining different materials such as polymers, cells, and specific architectures to assist regeneration of the injured tissue. This work objective is the conceptualization and manufacture of a multilayer multifunctional nanocomposite and study its cytocompatibility with neural stem cells (NSCs) envisioning NTE application. For that, vertically aligned carbon nanotubes (VACNTs) were grown by thermal chemical vapor deposition onto a silica substrate (SiO₂). Then, as-grown VACNTs were transferred onto polydimethylsiloxane (PDMS) creating a VACNT-PDMS layer with a stable interface, maintaining CNT alignment, confirmed by scanning electron microscopy. By transferring VACNTs onto this PDMS layer, flexibility and handling of the nanocomposite was obtained eliminating some problems presented by native VACNTs when coupled with their native substrate which is rigid. However, the nanocomposite VACNT/PDMS is inert and requires a component that mimics the neural microenvironment. So, a third polymeric layer was needed. To integrate this layer. VACNT and PDMS underwent a series of surface treatments (i.e., ultraviolet coupled with ozone (UV/O3) and oxygen (O₂) plasma) leading to changes on wettability and surface energy assessed indirectly by water contact angle (WCA) and attenuated-total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) was used to identify chemical changes post treatments. O₂ plasma treatment led to most significative change in wettability with the introduction of silanol (Si-OH) groups on PDMS and carboxylic (COOH) groups on CNTs. For the third layer two strategies were employed. The first (S1) relied on the creation of a hydrogel composed of gelatin and alginate methacrylamide (GelMA and AlgMA) capable of photo- and -ionic crosslink. The hydrogel showed to support NSC proliferation and viability, however, when transferred to the VACNT-PDMS layer the hydrogel would delaminate. The alternative solution (S2) consisted in the crosslink of gelatin and alginate induced by 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide coupled with N-hydroxysuccinimide on top of a previously silanized VACNT-PDMS layer. This was a successful approach creating a nanocomposite capable of sustaining adhesion and proliferation of NSCs. Furthermore, with immunocytochemical staining it was possible to observe neuronal differentiation. The results obtained demonstrated that the conceptualized multilayer multifunctional nanocomposite favors proliferation and differentiation of NSCs, making it an electro-responsive platform and a candidate to be electrically stimulated for neural tissue engineering.

In this study, alginate nanocomposite hydrogel bioinks reinforced with lysozyme nanofibers (LNFs) were developed. Alginate-LNF (A-LNF) suspensions with different LNF contents (1, 5 and 10 wt.%) were prepared and pre-crosslinked with 0.5% (w/v) CaCl2 to formulate A-LNF inks. These inks exhibit proper shear-thinning behavior and good recovery properties (~90%), with the pre-crosslinking step playing a crucial role. A-LNF fully crosslinked hydrogels (with 2% (w/v) CaCl2) that mimic 3D printing scaffolds were prepared, and it was observed that the addition of LNFs improved several properties of the hydrogels, such as the morphology, swelling and degradation profiles, and mechanical properties. All formulations are also noncytotoxic towards HaCaT cells. The printing parameters and 3D scaffold model were then optimized, with A-LNF inks showing improved printability. Selected A-LNF inks (A-LNF0 and A-LNF5) were loaded with HaCaT cells (cell density 2 × 106 cells mL-1), and the cell viability within the bioprinted scaffolds was evaluated for 1, 3 and 7 days, with scaffolds printed with the A-LNF5 bioink showing the highest values for 7 days (87.99 ± 1.28%). Hence, A-LNF bioinks exhibited improved rheological performance, printability and biological properties representing a good strategy to overcome the main limitations of alginate-based bioinks.

The development of suitable bioinks is an important research topic in the field of three-dimensional (3D) bioprinting. Herein, novel hydrogel-based bioinks composed of nanofibrillated cellulose (NFC) and gellan gum (GG) in different NFC/GG mass proportions (90:10, 80:20, 70:30, and 60:40) were developed and characterized. The increase in the content of GG, as well as its combination with NFC, enhanced their rheological properties, increasing both storage (G') and loss (G") moduli and the G' recovery capacity of the hydrogels (from 70.05 ± 3.06 % (90:10) to 82.63 ± 1.21 % (60:40)), as well as their mechanical properties, increasing the compressive stiffness and stress from 114.02 ± 10.93 Pa (90:10) to 337.16 ± 34.03 Pa (60:40) and from 18.27 ± 1.32 kPa (90:10) to 47.17 ± 3.59 kPa (60:40), respectively. The hydrogels were non-cytotoxic against human keratinocyte cells (HaCaT), with cell viabilities above 70 % for up to 72 h. The hydrogel 60:40 was loaded with HaCaT cells (3 × 106 cells mL-1) and bioprinted. The cell viability was maintained elevated until day 7 (90 ± 3 %) after bioprinting. These results highlight that the combination of these two biopolymers was a good strategy for the development of novel hydrogel-based bioinks for extrusion 3D bioprinting applications.