Therapeutic Opportunities and Delivery Strategies for Brain Revascularization in Stroke, Neurodegeneration, and Aging.

Central nervous system (CNS) diseases, especially acute ischemic events and neurodegenerative disorders, constitute a public health problem with no effective treatments to allow a persistent solution. Failed therapies targeting neuronal recovery have revealed the multifactorial and intricate pathophysiology underlying such CNS disorders as ischemic stroke, Alzheimers disease, amyotrophic lateral sclerosis, vascular Parkisonism, vascular dementia, and aging, in which cerebral microvasculature impairment seems to play a key role. In fact, a reduction in vessel density and cerebral blood flow occurs in these scenarios, contributing to neuronal dysfunction and leading to loss of cognitive function. In this review, we provide an overview of healthy brain microvasculature structure and function in health and the effect of the aforementioned cerebral CNS diseases. We discuss the emerging new therapeutic opportunities, and their delivery approaches, aimed at recovering brain vascularization in this context. SIGNIFICANCE STATEMENT: The lack of effective treatments, mainly focused on neuron recovery, has prompted the search of other therapies to treat cerebral central nervous system diseases. The disruption and degeneration of cerebral microvasculature has been evidenced in neurodegenerative diseases, stroke, and aging, constituting a potential target for restoring vascularization, neuronal functioning, and cognitive capacities by the development of therapeutic pro-angiogenic strategies.

Decellularized Extracellular Matrix-Based Bioinks for Tendon Regeneration in Three-Dimensional Bioprinting.

In the last few years, attempts to improve the regeneration of damaged tendons have been rising due to the growing demand. However, current treatments to restore the original performance of the tissue focus on the usage of grafts; although, actual grafts are deficient because they often cannot provide enough support for tissue regeneration, leading to additional complications. The beneficial effect of combining 3D bioprinting and dECM as a novel bioink biomaterial has recently been described. Tendon dECMs have been obtained by using either chemical, biological, or/and physical treatments. Although decellularization protocols are not yet standardized, recently, different protocols have been published. New therapeutic approaches embrace the use of dECM in bioinks for 3D bioprinting, as it has shown promising results in mimicking the composition and the structure of the tissue. However, major obstacles include the poor structural integrity and slow gelation properties of dECM bioinks. Moreover, printing parameters such as speed and temperature have to be optimized for each dECM bioink. Here, we show that dECM bioink for 3D bioprinting provides a promising approach for tendon regeneration for future clinical applications.

Hyaluronic Acid Promotes Differentiation of Mesenchymal Stem Cells from Different Sources toward Pancreatic Progenitors within Three-Dimensional Alginate Matrixes.

Islet transplantation has shown to be a successful alternative in type 1 diabetes treatment, but donor scarcity precludes its worldwide clinical translation. Stem cells are an unlimited source that could circumvent the lack of donors if complete differentiation into insulin-producing cells (IPCs) could be accomplished. We have performed the differentiation of mesenchymal stem cells (MSCs) from different sources into IPCs within three-dimensional (3D) alginate matrixes. We quantified an increased insulin release at the final stage of differentiation compared to undifferentiated MSCs, which is more pronounced in IPCs differentiated from pancreatic-derived MSCs tissues. Moreover, the addition of hyaluronic acid (HA) in alginate microcapsules enhanced, even more, the insulin release from the final IPCs, independent of the MSC source. We can conclude that MSCs can be differentiated into IPCs within alginate microcapsules, enhancing insulin release when HA is present in the 3D alginate matrixes.

Alginate Microcapsules Incorporating Hyaluronic Acid Recreate Closer in Vivo Environment for Mesenchymal Stem Cells.

The potential clinical application of alginate cell microencapsulation has advanced enormously during the past decade. However, the 3D environment created by alginate beads does not mimic the natural extracellular matrix surrounding cells in vivo, responsible of cell survival and functionality. As one of the most frequent macromolecules present in the extracellular matrix is hyaluronic acid, we have formed hybrid beads with alginate and hyaluronic acid recreating a closer in vivo cell environment. Our results show that 1% alginate-0.25% hyaluronic acid microcapsules retain 1.5% alginate physicochemical properties. Moreover, mesenchymal stem cells encapsulated in these hybrid beads show enhanced viability therapeutic protein release and mesenchymal stem cells' potential to differentiate into chondrogenic lineage. Although future studies with additional proteins need to be done in order to approach even more the extracellular matrix features, we have shown that hyaluronic acid protects alginate encapsulated mesenchymal stem cells by providing a niche-like environment and remaining them competent as a sustainable drug delivery system.

Hybrid Alginate-Protein-Coated Graphene Oxide Microcapsules Enhance the Functionality of Erythropoietin Secreting C2C12 Myoblasts.

The beneficial effect of combining alginate hydrogel with graphene oxide (GO) on microencapsulated C2C12-myoblast viability has recently been described. However, the commercially available GO lacks homogeneity in size, this parameter being of high relevance for the cell fate in two-dimensional studies. In three-dimensional applications the capacity of this material for binding different kinds of proteins can result in the reduction of de novo released protein that can effectively reach the vicinity of the microcapsules. Undoubtedly, this could be an important hurdle in its clinical use when combined with alginate-PLL microcapsules. Here, we demonstrate that the homogenization of GO nanoparticles is not a mandatory preparation step in order to get the best of this material upon cell microencapsulation. In fact, when the superficial area of these particles is increased, higher amounts of the therapeutic protein erythropoietin (EPO) are adsorbed on their surface. On the other hand, we have been able to improve even more the favorable effects of this graphene derivative on microencapsulated cell viability by forming a protein biocorona. These proteins block the potential binding sites of EPO and, therefore, enhance the amount of therapeutic drug that is released. Finally, we prove that these hybrid alginate-protein-coated GO-microcapsules are functional in vivo.

Nanotherapeutic approaches for brain cancer management.

UNLABELLED: Around the world, cancer remains one of the most important causes of morbidity and mortality. Worldwide, approximately 238,000 new cases of brain and other central nervous system tumors are diagnosed every year. Nanotherapeutic approaches hold tremendous potential for diagnosis and treatment of brain cancer, including the ability to target complex molecular cargoes to the tumor sites and the capacity of crossing the blood-brain barrier and accessing to the brain after systemic administration. A new generation of "smart" nanoparticles has been designed as novel targeted delivery devices for new therapies including gene therapy, anti-angiogenic and thermotherapy. This review highlights the latest research, opportunities and challenges for developing novel nanotherapeutics for treating brain cancers. FROM THE CLINICAL EDITOR: This comprehensive review highlights the latest research results, opportunities and challenges for developing novel nanotherapeutics for treating brain cancers, with a special focus on "smart" nanoparticles as novel targeted delivery devices for new therapies including gene therapy, anti-angiogenic therapy and localized thermotherapy.

The role of microfluidics and 3D-bioprinting in the future of exosome therapy.

Exosome-based strategies constitute a promising tool for therapeutics, avoiding potential immunogenic and tumorigenic side-effects of cell therapies. However, the collection of a suitable exosome pool, and the need for high doses with conventional administration approaches, hamper their clinical translation. To overcome these challenges, versatile exosome collection strategies together with advanced delivery platforms may represent major progress in this field. Microfluidics enables large-scale gathering of both natural and synthetic exosomes for their implementation into bioinks, while 3D-bioprinting holds great promise in regenerative medicine with the use of exosome-loaded scaffolds that mimic the target tissue with controlled pharmacokinetics and pharmacodynamics. Hence, the combination of both strategies might become the key for the translation of exosome therapies to clinical practice.

Stability of Monoclonal Antibodies as Solid Formulation for Auto-Injectors: A Pilot Study.

Drug adherence is a significant medical issue, often responsible for sub-optimal outcomes during the treatment of chronic diseases such as rheumatoid or psoriatic arthritis. Monoclonal antibodies (which are exclusively given parenterally) have been proven to be an effective treatment in these cases. The use of auto-injectors is an effective strategy to improve drug adherence in parenteral treatments since these pen-like devices offer less discomfort and increased user-friendliness over conventional syringe-based delivery. This study aims to investigate the feasibility of including a monoclonal antibody as a solid formulation inside an auto-injector pen. Specifically, the objective was to evaluate the drug stability after a concentration (to reduce the amount of solvent and space needed) and freeze-drying procedure. A preliminary screening of excipients to improve stability was also performed. The nano-DSC results showed that mannitol improved the stability of the concentrated, freeze-dried antibody in comparison to its counterpart without it. However, a small instability of the CH2 domain was still found for mannitol samples, which will warrant further investigation. The present results serve as a stepping stone towards advancing future drug delivery systems that will ultimately improve the patient experience and associated drug adherence.

219Three-dimensional printing as a cutting-edge, versatile and personalizable vascular stent manufacturing procedure: Toward tailor-made medical devices.

Vascular stents (VS) have revolutionized the treatment of cardiovascular diseases, as evidenced by the fact that the implantation of VS in coronary artery disease (CAD) patients has become a routine, easily approachable surgical intervention for the treatment of stenosed blood vessels. Despite the evolution of VS throughout the years, more efficient approaches are still required to address the medical and scientific challenges, especially when it comes to peripheral artery disease (PAD). In this regard, three-dimensional (3D) printing is envisaged as a promising alternative to upgrade VS by optimizing the shape, dimensions and stent backbone (crucial for optimal mechanical properties), making them customizable for each patient and each stenosed lesion. Moreover, the combination of 3D printing with other methods could also upgrade the final device. This review focuses on the most recent studies using 3D printing techniques to produce VS, both by itself and in combination with other techniques. The final aim is to provide an overview of the possibilities and limitations of 3D printing in the manufacturing of VS. Furthermore, the current situation of CAD and PAD pathologies is also addressed, thus highlighting the main weaknesses of the already existing VS and identifying research gaps, possible market niches and future directions.

Characterization and assessment of new fibrillar collagen inks and bioinks for 3D printing and bioprinting.

Collagen is a cornerstone protein for tissue engineering and 3D bioprinting due to its outstanding biocompatibility, low immunogenicity, and natural abundance in human tissues. Nonetheless, it still poses some important challenges, such as complicated and limited extraction processes, usually accompanied by batch- to-batch reproducibility and influence of factors, such as temperature, pH, and ionic strength. In this work, we evaluated the suitability and performance of new, fibrillar type I collagen as standardized and reproducible collagen source for 3D printing and bioprinting. The acidic, native fibrous collagen formulation (5% w/w) performed remarkably during 3D printing, which was possible to print constructs of up to 27 layers without collapsing. On the other hand, the fibrous collagen mass has been modified to provide a fast, reliable, and easily neutralizable process. The neutralization with TRIS-HCl enabled the inclusion of cells without hindering printability. The cell-laden constructs were printed under mild conditions (50-80 kPa, pneumatic 3D printing), providing remarkable cellular viability (>90%) as well as a stable platform for cell growth and proliferation in vitro. Therefore, the native, type I collagen masses characterized in this work offer a reproducible and reliable source of collagen for 3D printing and bioprinting purposes.

Progress in 3D Bioprinting Technology for Osteochondral Regeneration.

Osteochondral injuries can lead to osteoarthritis (OA). OA is characterized by the progressive degradation of the cartilage tissue together with bone tissue turnover. Consequently, joint pain, inflammation, and stiffness are common, with joint immobility and dysfunction being the most severe symptoms. The increase in the age of the population, along with the increase in risk factors such as obesity, has led OA to the forefront of disabling diseases. In addition, it not only has an increasing prevalence, but is also an economic burden for health systems. Current treatments are focused on relieving pain and inflammation, but they become ineffective as the disease progresses. Therefore, new therapeutic approaches, such as tissue engineering and 3D bioprinting, have emerged. In this review, the advantages of using 3D bioprinting techniques for osteochondral regeneration are described. Furthermore, the biomaterials, cell types, and active molecules that are commonly used for these purposes are indicated. Finally, the most recent promising results for the regeneration of cartilage, bone, and/or the osteochondral unit through 3D bioprinting technologies are considered, as this could be a feasible therapeutic approach to the treatment of OA.

Chondroitin and Dermatan Sulfate Bioinks for 3D Bioprinting and Cartilage Regeneration.

Cartilage is a connective tissue which a limited capacity for healing and repairing. In this context, osteoarthritis (OA) disease may be developed with high prevalence in which the use of scaffolds may be a promising treatment. In addition, three-dimensional (3D) bioprinting has become an emerging additive manufacturing technology because of its rapid prototyping capacity and the possibility of creating complex structures. This study is focused on the development of nanocellulose-alginate (NC-Alg) based bioinks for 3D bioprinting for cartilage regeneration to which it is added chondroitin sulfate (CS) and dermatan sulfate (DS). First, rheological properties are evaluated. Then, sterilization effect, biocompatibility, and printability on developed NC-Alg-CS and NC-Alg-DS inks are evaluated. Subsequently, printed scaffolds are characterized. Finally, NC-Alg-CS and NC-Alg-DS inks are loaded with murine D1-MSCs-EPO and cell viability and functionality, as well as the chondrogenic differentiation ability are assessed. Results show that the addition of both CS and DS to the NC-Alg ink improves its characteristics in terms of rheology and cell viability and functionality. Moreover, differentiation to cartilage is promoted on NC-Alg-CS and NC-Alg-DS scaffolds. Therefore, the utilization of MSCs containing NC-Alg-CS and NC-Alg-DS scaffolds may become a feasible tissue engineering approach for cartilage regeneration.

3D Bioprinted Hydroxyapatite or Graphene Oxide Containing Nanocellulose-Based Scaffolds for Bone Regeneration.

Bone tissue is usually damaged after big traumas, tumors, and increasing aging-related diseases such as osteoporosis and osteoarthritis. Current treatments are based on implanting grafts, which are shown to have several inconveniences. In this regard, tissue engineering through the 3D bioprinting technique has arisen to manufacture structures that would be a feasible therapeutic option for bone regenerative medicine. In this study, nanocellulose-alginate (NC-Alg)-based bioink is improved by adding two different inorganic components such as hydroxyapatite (HAP) and graphene oxide (GO). First, ink rheological properties and biocompatibility are evaluated as well as the influence of the sterilization process on them. Then, scaffolds are characterized. Finally, biological studies of embedded murine D1 mesenchymal stem cells engineered to secrete erythropoietin are performed. Results show that the addition of both HAP and GO prevents NC-Alg ink from viscosity lost in the sterilization process. However, GO is reduced due to short cycle autoclave sterilization, making it incompatible with this ink. In addition, HAP and GO have different influences on scaffold architecture and surface as well as in swelling capacity. Scaffolds mechanics, as well as cell viability and functionality, are promoted by both elements addition. Additionally, GO demonstrates an enhanced bone differentiation capacity.

Tendon tissue engineering: Cells, growth factors, scaffolds and production techniques.

Tendon injuries are a global health problem that affects millions of people annually. The properties of tendons make their natural rehabilitation a very complex and long-lasting process. Thanks to the development of the fields of biomaterials, bioengineering and cell biology, a new discipline has emerged, tissue engineering. Within this discipline, diverse approaches have been proposed. The obtained results turn out to be promising, as increasingly more complex and natural tendon-like structures are obtained. In this review, the nature of the tendon and the conventional treatments that have been applied so far are underlined. Then, a comparison between the different tendon tissue engineering approaches that have been proposed to date is made, focusing on each of the elements necessary to obtain the structures that allow adequate regeneration of the tendon: growth factors, cells, scaffolds and techniques for scaffold development. The analysis of all these aspects allows understanding, in a global way, the effect that each element used in the regeneration of the tendon has and, thus, clarify the possible future approaches by making new combinations of materials, designs, cells and bioactive molecules to achieve a personalized regeneration of a functional tendon.

Current Insights Into 3D Bioprinting: An Advanced Approach for Eye Tissue Regeneration.

Three-dimensional (3D) printing is a game changer technology that holds great promise for a wide variety of biomedical applications, including ophthalmology. Through this emerging technique, specific eye tissues can be custom-fabricated in a flexible and automated way, incorporating different cell types and biomaterials in precise anatomical 3D geometries. However, and despite the great progress and possibilities generated in recent years, there are still challenges to overcome that jeopardize its clinical application in regular practice. The main goal of this review is to provide an in-depth understanding of the current status and implementation of 3D bioprinting technology in the ophthalmology field in order to manufacture relevant tissues such as cornea, retina and conjunctiva. Special attention is paid to the description of the most commonly employed bioprinting methods, and the most relevant eye tissue engineering studies performed by 3D bioprinting technology at preclinical level. In addition, other relevant issues related to use of 3D bioprinting for ocular drug delivery, as well as both ethical and regulatory aspects, are analyzed. Through this review, we aim to raise awareness among the research community and report recent advances and future directions in order to apply this advanced therapy in the eye tissue regeneration field.

Clay Minerals as Bioink Ingredients for 3D Printing and 3D Bioprinting: Application in Tissue Engineering and Regenerative Medicine.

The adaptation and progress of 3D printing technology toward 3D bioprinting (specifically adapted to biomedical purposes) has opened the door to a world of new opportunities and possibilities in tissue engineering and regenerative medicine. In this regard, 3D bioprinting allows for the production of tailor-made constructs and organs as well as the production of custom implants and medical devices. As it is a growing field of study, currently, the attention is heeded on the optimization and improvement of the mechanical and biological properties of the so-called bioinks/biomaterial inks. One of the strategies proposed is the use of inorganic ingredients (clays, hydroxyapatite, graphene, carbon nanotubes and other silicate nanoparticles). Clays have proven to be useful as rheological and mechanical reinforcement in a wide range of fields, from the building industry to pharmacy. Moreover, they are naturally occurring materials with recognized biocompatibility and bioactivity, revealing them as optimal candidates for this cutting-edge technology. This review deals with the use of clays (both natural and synthetic) for tissue engineering and regenerative medicine through 3D printing and bioprinting. Despite the limited number of studies, it is possible to conclude that clays play a fundamental role in the formulation and optimization of bioinks and biomaterial inks since they are able to improve their rheology and mechanical properties, thus improving printability and construct resistance. Additionally, they have also proven to be exceptionally functional ingredients (enhancing cellular proliferation, adhesion, differentiation and alignment), controlling biodegradation and carrying/releasing actives with tissue regeneration therapeutic activities.

Characterization of encapsulated porcine cardiosphere-derived cells embedded in 3D alginate matrices.

Myocardial infarction is caused by an interruption of coronary blood flow, leading to one of the main death causes worldwide. Current therapeutic approaches are palliative and not able to solve the loss of cardiac tissue. Cardiosphere derived cells (CDCs) reduce scarring, and increase viable myocardium, with safety and adequate biodistribution, but show a low rate engraftment and survival after implantation. In order to solve the low retention, we propose the encapsulation of CDCs within three-dimensional alginate-poly-L-lysine-alginate matrix as therapy for cardiac regeneration. In this work, we demonstrate the encapsulation of CDCs in alginate matrix, with no decrease in viability over a month, and showing the preservation of CDCs phenotype, differentiation potential, gene expression profile and growth factor release after encapsulation, moving a step forward to clinical translation of CDCs therapy in regeneration in heart failure.

Modulation of Conductivity of Alginate Hydrogels Containing Reduced Graphene Oxide through the Addition of Proteins.

Modifying hydrogels in order to enhance their conductivity is an exciting field with applications in cardio and neuro-regenerative medicine. Therefore, we have designed hybrid alginate hydrogels containing uncoated and protein-coated reduced graphene oxide (rGO). We specifically studied the adsorption of three different proteins, BSA, elastin, and collagen, and the outcomes when these protein-coated rGO nanocomposites are embedded within the hydrogels. Our results demonstrate that BSA, elastin, and collagen are adsorbed onto the rGO surface, through a non-spontaneous phenomenon that fits Langmuir and pseudo-second-order adsorption models. Protein-coated rGOs are able to preclude further adsorption of erythropoietin, but not insulin. Collagen showed better adsorption capacity than BSA and elastin due to its hydrophobic nature, although requiring more energy. Moreover, collagen-coated rGO hybrid alginate hydrogels showed an enhancement in conductivity, showing that it could be a promising conductive scaffold for regenerative medicine.

Force Spectroscopy Imaging and Constriction Assays Reveal the Effects of Graphene Oxide on the Mechanical Properties of Alginate Microcapsules.

Microencapsulation of cells in hydrogel-based porous matrices is an approach that has demonstrated great success in regenerative cell therapy. These microcapsules work by concealing the exogenous cells and materials in a robust biomaterial that prevents their recognition by the immune system. A vast number of formulations and additives are continuously being tested to optimize cell viability and mechanical properties of the hydrogel. Determining the effects of new microcapsule additives is a lengthy process that usually requires extensive in vitro and in vivo testing. In this paper, we developed a workflow using nanoindentation (i.e., indentation with a nanoprobe in an atomic force microscope) and a custom-built microfluidic constriction device to characterize the effect of graphene oxide (GO) on three microcapsule formulations. With our workflow, we determined that GO modifies the microcapsule stiffness and surface properties in a formulation-dependent manner. Our results also suggest, for the first time, that GO alters the conformation of the microcapsule hydrogel and its interaction with subsequent coatings. Overall, our workflow can infer the effects of new additives on microcapsule surfaces. Thus, our workflow can contribute to diminishing the time required for the validation of new microcapsule formulations and accelerate their clinical translation.

Graphene oxide and reduced graphene oxide-based scaffolds in regenerative medicine.

There is a vast and rapid increase in the applications of graphene oxide (GO) and reduced graphene oxide (rGO) in the biomedical field, including drug delivery, bio-sensing, and diagnostic tools. Among all the applications, the GO and rGO-based scaffolds are a very promising system that have attracted attention because of their great clinical projection in tissue regeneration therapies. Both GO and rGO have shown a strong impact on the proliferation and differentiation of implemented stem cells, but still need to overcome several challenges, such as cytotoxicity, biodistribution, biotransformation or immune response. However, there are still controversial hypothesises regarding the mechanisms involved in these issues that should be clarified in order to improve the applications of these compounds. 3D-scaffolds can help in solving some of those limitations when moving into preclinical studies in regenerative medicine. In this review, we will describe the application of GO and rGO within 3D scaffolds in bone, cardiac and neural regenerative medicine after analyzing the aforementioned challenges.