Hierarchical Design of Tissue-Mimetic Fibrillar Hydrogel Scaffolds.

Most tissues of the human body present hierarchical fibrillar extracellular matrices (ECMs) that have a strong influence over their physicochemical properties and biological behavior. Of great interest is the introduction of this fibrillar structure to hydrogels, particularly due to the water-rich composition, cytocompatibility, and tunable properties of this class of biomaterials. Here, the main bottom-up fabrication strategies for the design and production of hierarchical biomimetic fibrillar hydrogels and their most representative applications in the fields of tissue engineering and regenerative medicine are reviewed. For example, the controlled assembly/arrangement of peptides, polymeric micelles, cellulose nanoparticles (NPs), and magnetically responsive nanostructures, among others, into fibrillar hydrogels is discussed, as well as their potential use as fibrillar-like hydrogels (e.g., those from cellulose NPs) with key biofunctionalities such as electrical conductivity or remote stimulation. Finally, the major remaining barriers to the clinical translation of fibrillar hydrogels and potential future directions of research in this field are discussed.

A dive into the bath: embedded 3D bioprinting of freeform in vitro models.

Designing functional, vascularized, human scale in vitro models with biomimetic architectures and multiple cell types is a highly promising strategy for both a better understanding of natural tissue/organ development stages to inspire regenerative medicine, and to test novel therapeutics on personalized microphysiological systems. Extrusion-based 3D bioprinting is an effective biofabrication technology to engineer living constructs with predefined geometries and cell patterns. However, bioprinting high-resolution multilayered structures with mechanically weak hydrogel bioinks is challenging. The advent of embedded 3D bioprinting systems in recent years offered new avenues to explore this technology for in vitro modeling. By providing a stable, cell-friendly and perfusable environment to hold the bioink during and after printing, it allows to recapitulate native tissues' architecture and function in a well-controlled manner. Besides enabling freeform bioprinting of constructs with complex spatial organization, support baths can further provide functional housing systems for their long-term in vitro maintenance and screening. This minireview summarizes the recent advances in this field and discuss the enormous potential of embedded 3D bioprinting technologies as alternatives for the automated fabrication of more biomimetic in vitro models.

Central Venous Catheter-Associated Superior Vena Cava Syndrome.

Superior vena cava syndrome (SVCS) is caused by any obstruction to the superior vena cava (SVC); the most common causes are malignancy and extrinsic compression. The use of medical devices, such as central venous catheters, poses an important risk factor, as they cause changes in the blood flow and in the vessel wall. This report describes a case of a 70-year-old male with an implantable central venous port, due to previous neoplastic disease, as the cause of the SVCS. Authors advise that the placement of medical devices ought to be carefully accessed and constantly revised to be removed when no longer needed to prevent avoidable complications.

Writing 3D In Vitro Models of Human Tendon within a Biomimetic Fibrillar Support Platform.

Tendinopathies are poorly understood diseases for which treatment remains challenging. Relevant in vitro models to study human tendon physiology and pathophysiology are therefore highly needed. Here we propose the automated 3D writing of tendon microphysiological systems (MPSs) embedded in a biomimetic fibrillar support platform based on cellulose nanocrystals (CNCs) self-assembly. Tendon decellularized extracellular matrix (dECM) was used to formulate bioinks that closely recapitulate the biochemical signature of tendon niche. A monoculture system recreating the cellular patterns and phenotype of the tendon core was first developed and characterized. This system was then incorporated with a vascular compartment to study the crosstalk between the two cell populations. The combined biophysical and biochemical cues of the printed pattern and dECM hydrogel were revealed to be effective in inducing human-adipose-derived stem cells (hASCs) differentiation toward the tenogenic lineage. In the multicellular system, chemotactic effects promoted endothelial cells migration toward the direction of the tendon core compartment, while the established cellular crosstalk boosted hASCs tenogenesis, emulating the tendon development stages. Overall, the proposed concept is a promising strategy for the automated fabrication of humanized organotypic tendon-on-chip models that will be a valuable new tool for the study of tendon physiology and pathogenesis mechanisms and for testing new tendinopathy treatments.

Platelet-Derived Extracellular Vesicles Promote Tenogenic Differentiation of Stem Cells on Bioengineered Living Fibers.

Tendon mimetic scaffolds that recreate the tendon hierarchical structure and niche have increasing potential to fully restore tendon functionality. However, most scaffolds lack biofunctionality to boost the tenogenic differentiation of stem cells. In this study, we assessed the role of platelet-derived extracellular vesicles (EVs) in stem cells' tenogenic commitment using a 3D bioengineered in vitro tendon model. First, we relied on fibrous scaffolds coated with collagen hydrogels encapsulating human adipose-derived stem cells (hASCs) to bioengineer our composite living fibers. We found that the hASCs in our fibers showed high elongation and cytoskeleton anisotropic organization, typical of tenocytes. Moreover, acting as biological cues, platelet-derived EVs boosted the hASCs' tenogenic commitment, prevented phenotypic drift, enhanced the deposition of the tendon-like extracellular matrix, and induced lower collagen matrix contraction. In conclusion, our living fibers provided an in vitro system for tendon tissue engineering, allowing us to study not only the tendon microenvironment but also the influence of biochemical cues on stem cell behavior. More importantly, we showed that platelet-derived EVs are a promising biochemical tool for tissue engineering and regenerative medicine applications that are worthy of further exploration, as paracrine signaling might potentiate tendon repair and regeneration.

Topographical and Compositional Gradient Tubular Scaffold for Bone to Tendon Interface Regeneration.

The enthesis is an extremely specific region, localized at the tendon-bone interface (TBI) and made of a hybrid connection of fibrocartilage with minerals. The direct type of enthesis tissue is commonly subjected to full laceration, due to the stiffness gradient between the soft tissues and hard bone, and this often reoccurs after surgical reconstruction. For this purpose, the present work aimed to design and develop a tubular scaffold based on pullulan (PU) and chitosan (CH) and intended to enhance enthesis repair. The scaffold was designed with a topographical gradient of nanofibers, from random to aligned, and hydroxyapatite (HAP) nanoparticles along the tubular length. In particular, one part of the tubular scaffold was characterized by a structure similar to bone hard tissue, with a random mineralized fiber arrangement; while the other part was characterized by aligned fibers, without HAP doping. The tubular shape of the scaffold was also designed to be extemporarily loaded with chondroitin sulfate (CS), a glycosaminoglycan effective in wound healing, before the surgery. Micro CT analysis revealed that the scaffold was characterized by a continuous gradient, without interruptions from one end to the other. The gradient of the fiber arrangement was observed using SEM analysis, and it was still possible to observe the gradient when the scaffold had been hydrated for 6 days. In vitro studies demonstrated that human adipose stem cells (hASC) were able to grow and differentiate onto the scaffold, expressing the typical ECM production for tendon in the aligned zone, or bone tissue in the random mineralized part. CS resulted in a synergistic effect, favoring cell adhesion/proliferation on the scaffold surface. These results suggest that this tubular scaffold loaded with CS could be a powerful tool to support enthesis repair upon surgery.

Heart Failure: From Typical Clinical Manifestations to the Surprising Final Diagnosis.

The authors report a case of an 80-year-old woman with multiple cardiovascular risk factors, with exuberant acute congestive heart failure at admission. Fever, anemia, and an increase in inflammatory parameters were present, with imaging suggesting a respiratory infection as the main reason for decompensation. Empirical antibiotic therapy was instituted, with no clinical improvement even after escalation to broad-spectrum antibiotics and non-invasive ventilation with high support pressures, with no possibility of weaning. Due to maintenance of symptoms, a transthoracic echocardiogram was performed, revealing a large left atrial myxoma, obstructing the mitral valve in diastole. This case illustrates the potential severity of these benign tumors and their ability to mimic symptoms that are often evaluated in the daily life of an internist. The high clinical suspicion led to a diagnosis that was surprising due to its rarity and severity, with the patient being urgently referred for cardiac surgery.

Xeno-free bioengineered human skeletal muscle tissue using human platelet lysate-based hydrogels.

Bioengineered human skeletal muscle tissues have emerged in the last years as newin vitrosystems for disease modeling. These bioartificial muscles are classically fabricated by encapsulating human myogenic precursor cells in a hydrogel scaffold that resembles the extracellular matrix. However, most of these hydrogels are derived from xenogenic sources, and the culture media is supplemented with animal serum, which could interfere in drug testing assays. On the contrary, xeno-free biomaterials and culture conditions in tissue engineering offer increased relevance for developing human disease models. In this work, we used human platelet lysate (PL)-based nanocomposite hydrogels (HUgel) as scaffolds for human skeletal muscle tissue engineering. These hydrogels consist of human PL reinforced with aldehyde-cellulose nanocrystals (a-CNC) that allow tunable mechanical, structural, and biochemical properties for the 3D culture of stem cells. Here, we developed hydrogel casting platforms to encapsulate human muscle satellite stem cells in HUgel. The a-CNC content was modulated to enhance matrix remodeling, uniaxial tension, and self-organization of the cells, resulting in the formation of highly aligned, long myotubes expressing sarcomeric proteins. Moreover, the bioengineered human muscles were subjected to electrical stimulation, and the exerted contractile forces were measured in a non-invasive manner. Overall, our results demonstrated that the bioengineered human skeletal muscles could be built in xeno-free cell culture platforms to assess tissue functionality, which is promising for drug development applications.

Highly elastic and bioactive bone biomimetic scaffolds based on platelet lysate and biomineralized cellulose nanocrystals.

Bone is a vascularized organic-inorganic composite tissue that shows a heavily-mineralized extracellular matrix (ECM) on the nanoscale. Herein, the nucleation of calcium phosphates during the biomineralization process was mimicked using negatively-charged cellulose nanocrystals (CNCs). These mineralized-CNCs were combined with platelet lysate to produce nanocomposite scaffolds through cryogelation to mimic bone ECM protein-mineral composite nature and take advantage of the bioactivity steaming from platelet-derived biomolecules. The nanocomposite scaffolds showed high microporosity (94-95%), high elasticity (recover from 75% strain cycles), injectability, and modulated platelet-derived growth factors sequestration and release. Furthermore, they increased alkaline phosphatase activity (up to 10-fold) and up-regulated the expression of bone-related markers (up to 2-fold), without osteogenic supplementation, demonstrating their osteoinductive properties. Also, the scaffolds promoted the chemotaxis of endothelial cells and enhanced the expression of endothelial markers, showing proangiogenic potential. These results suggest that the mineralized nanocomposite scaffolds can enhance bone regeneration by simultaneously promoting osteogenesis and angiogenesis.

Bioengineered 3D Living Fibers as In Vitro Human Tissue Models of Tendon Physiology and Pathology.

Clinically relevant in vitro models of human tissue's health and disease are urgently needed for a better understanding of biological mechanisms essential for the development of novel therapies. Herein, physiological (healthy) and pathological (disease) tendon states are bioengineered by coupling the biological signaling of platelet lysate components with controlled 3D architectures of electrospun microfibers to drive the fate of human tendon cells in different composite living fibers (CLFs). In the CLFs-healthy model, tendon cells adopt a high cytoskeleton alignment and elongation, express tendon-related markers (scleraxis, tenomodulin, and mohawk) and deposit a dense tenogenic matrix. In contrast, cell crowding with low preferential orientation, high matrix deposition, and phenotypic drift leading to increased expression of nontendon related and fibrotic markers, are characteristics of the CLFs-diseased model. This diseased-like profile, also reflected in the increase of COL3/COL1 ratio, is further evident by the imbalance between matrix remodeling and degradation effectors, characteristic of tendinopathy. In summary, microengineered 3D in vitro models of human tendon healthy and diseased states are successfully fabricated. Most importantly, these innovative and versatile microphysiological models offer major advantages over currently used systems, holding promise for drugs screening and development of new therapies.

The tendon microenvironment: Engineered in vitro models to study cellular crosstalk.

Tendinopathy is a multi-faceted pathology characterized by alterations in tendon microstructure, cellularity and collagen composition. Challenged by the possibility of regenerating pathological or ruptured tendons, the healing mechanisms of this tissue have been widely researched over the past decades. However, so far, most of the cellular players and processes influencing tendon repair remain unknown, which emphasizes the need for developing relevant in vitro models enabling to study the complex multicellular crosstalk occurring in tendon microenvironments. In this review, we critically discuss the insights on the interaction between tenocytes and the other tendon resident cells that have been devised through different types of existing in vitro models. Building on the generated knowledge, we stress the need for advanced models able to mimic the hierarchical architecture, cellularity and physiological signaling of tendon niche under dynamic culture conditions, along with the recreation of the integrated gradients of its tissue interfaces. In a forward-looking vision of the field, we discuss how the convergence of multiple bioengineering technologies can be leveraged as potential platforms to develop the next generation of relevant in vitro models that can contribute for a deeper fundamental knowledge to develop more effective treatments.

Controlling the fate of regenerative cells with engineered platelet-derived extracellular vesicles.

Extracellular vesicles (EVs) have emerged as cell-free nanotherapeutic agents for the potential treatment of multiple diseases and for tissue engineering and regenerative medicine strategies. Nevertheless, the field has typically relied on EVs derived from stem cells, the production of which in high quantities and high reproducibility is still under debate. Platelet-derived EVs were produced by a freeze-thaw method of platelet concentrates, a highly available clinical waste material. The aim of this study was to produce and thoroughly characterize platelet-derived EVs and understand their effects in adipose-tissue derived stem cells (hASCs), endothelial cells (HUVECs) and macrophages. Two different EV populations were obtained after differential centrifugation, namely small EVs (sEVs) and medium EVs (mEVs), which showed different size distributions and unique proteomic signatures. EV interaction with hASCs resulted in the modulation of the gene expression of markers related to their commitment toward different lineages. Moreover, mEVs showed higher angiogenic potential than sEVs, in a tube formation assay with HUVECs. Also, the EVs were able to modulate macrophage polarization. Altogether, these results suggest that platelet-derived EVs are promising candidates to be used as biochemical signals or therapeutic tools in tissue engineering and regenerative medicine approaches.

Therapeutic Effects of Platelet-Derived Extracellular Vesicles in a Bioengineered Tendon Disease Model.

Tendon injuries represent over 30-50% of musculoskeletal disorders worldwide, yet the available therapies do not provide complete tendon repair/regeneration and full functionality restoring. Extracellular vesicles (EVs), membrane-enclosed nanoparticles, have emerged as the next breakthrough in tissue engineering and regenerative medicine to promote endogenous tissue regeneration. Here, we developed a 3D human in vitro model mimicking the signature of pathological tendon and used it to evaluate the influence that different platelet-derived EVs might have in tendon tissue repair mechanisms. For this, different EV populations isolated from platelets, small EVs (sEVs) and medium EVs (mEVs), were added to the culture media of human tendon-derived cells (hTDCs) cultured on isotropic nanofibrous scaffolds. The platelet-derived EVs increased the expression of tenogenic markers, promoted a healthy extracellular matrix (ECM) remodeling, and the synthesis of anti-inflammatory mediators. These findings suggest that platelet EVs provided relevant biochemical cues that potentiated a recovery of hTDCs phenotype from a diseased to a healthy state. Thus, this study opens new perspectives for the translation of platelet-derived EVs as therapeutics.

Texturing Hierarchical Tissues by Gradient Assembling of Microengineered Platelet-Lysates Activated Fibers.

The heterogeneity of hierarchical tissues requires designing multipart engineered constructs as suitable tissue replacements. Herein, the incorporation of platelet lysate (PL) within an electrospun fiber core is proposed aiming for the fabrication of functionally graded 3D scaffolds for heterotypic tissues regeneration, such as tendon-to-bone interfaces. First, anisotropic yarns (A-Yarns) and isotropic threads with nanohydroxyapatite (I-Threads/PL@nHAp) are fabricated to recreate the tendon- and bone-microstructures and both incorporated with PL using emulsion electrospinning for a sustained and local delivery of growth factors, cytokines, and chemokines. Biological performance using human adipose-derived stem cells demonstrates that A-Yarns/PL induce a higher expression of scleraxis, a tenogenic-marker, while in I-Threads/PL@nHAp, higher alkaline phosphatase activity and matrix mineralization suggest an osteogenic commitment without the need for biochemical supplementation compared to controls. As a proof-of-concept, functional 3D gradient scaffolds are fabricated using a weaving technique, resulting in 3D textured hierarchical constructs with gradients in composition and topography. Additionally, the precise delivery of bioactive cues together with in situ biophysical features guide the commitment into a phenotypic gradient exhibiting chondrogenic and osteochondrogenic profiles in the interface of scaffolds. Overall, a promising patch solution for the regeneration of tendon-to-bone tissue interface through the fabrication of PL-functional 3D gradient constructs is demonstrated.

Epitope-imprinted polymers: Design principles of synthetic binding partners for natural biomacromolecules.

Molecular imprinting (MI) has been explored as an increasingly viable tool for molecular recognition in various fields. However, imprinting of biologically relevant molecules like proteins is severely hampered by several problems. Inspired by natural antibodies, the use of epitopes as imprinting templates has been explored to circumvent those limitations, offering lower costs and greater versatility. Here, we review the latest innovations in this technology, as well as different applications where MI polymers (MIPs) have been used to target biomolecules of interest. We discuss the several steps in MI, from the choice of epitope and functional monomers to the different production methods and possible applications. We also critically explore how MIP performance can be assessed by various parameters. Last, we present perspectives on future breakthroughs and advances, offering insights into how MI techniques can be expanded to new fields such as tissue engineering.

Human Platelet Lysate-Loaded Poly(ethylene glycol) Hydrogels Induce Stem Cell Chemotaxis In Vitro.

Platelet lysates (PL) contain a selection of proteins and growth factors (GFs) that are known to mediate cell activity. Many of these biomolecules have been identified as chemoattractants with the capacity to induce cell migration. In order to effectively deliver and retain these biomolecules to the site of injury, a scaffold containing PL could be an option. We use poly(ethylene glycol) (PEG) hydrogels consisting of 90 vol % PL to investigate their migratory potential on human mesenchymal stem cells (hMSCs). Cells exposed to these hydrogels were tracked, resulting in cell trajectories and detailed migratory parameters (velocity, Euclidean distance, directness, and forward migration index). Volumetric swelling ratios, hydrogel mechanical properties, and the release kinetics of proteins and GFs from hydrogels were also assessed. Furthermore, hMSC spheroids were encapsulated within the hydrogels to qualitatively assess cell invasion by means of sprouting and disintegration of the spheroid. Cell spheroids encapsulated within the PL-PEG gels exhibited initial outgrowths and eventually colonized the 3D matrix successfully. Results from this study confirmed that hMSCs exhibit directional migration toward the PL-loaded hydrogel with increased velocity and directness, compared to the controls. Overall, the incorporation of PL renders the PEG hydrogel bioactive. This study demonstrates the capacity of PL-loaded hydrogel constructs to attract stem cells for endogenous tissue engineering purposes.

Engineering next-generation bioinks with nanoparticles: moving from reinforcement fillers to multifunctional nanoelements.

The application of additive manufacturing in the biomedical field has become a hot topic in the last decade owing to its potential to provide personalized solutions for patients. Different bioinks have been designed trying to obtain a unique concoction that addresses all the needs for tissue engineering and drug delivery purposes, among others. Despite the remarkable progress made, the development of suitable bioinks which combine printability, cytocompatibility, and biofunctionality is still a challenge. In this sense, the well-established synthetic and functionalization routes to prepare nanoparticles with different functionalities make them excellent candidates to be combined with polymeric systems in order to generate suitable multi-functional bioinks. In this review, we briefly discuss the most recent advances in the design of functional nanocomposite hydrogels considering their already evaluated or potential use as bioinks. The scientific development over the last few years is reviewed, focusing the discussion on the wide range of functionalities that can be incorporated into 3D bioprinted constructs through the addition of multifunctional nanoparticles in order to increase their regenerative potential in the field of tissue engineering.

Multifunctional Surfaces for Improving Soft Tissue Integration.

Metallic implants are widely used in diverse clinical applications to aid in recovery from lesions or to replace native hard tissues. However, the lack of integration of metallic surfaces with soft tissue interfaces causes the occurrence of biomaterial-associated infections, which can trigger a complicated inflammatory response and, ultimately, implant failure. Here, a multifunctional implant surface showing nanoscale anisotropy, based on the controlled deposition of cellulose nanocrystals (CNC), and biological activity derived from platelet lysate (PL) biomolecules sequestered and presented on CNC surface, is proposed. The anisotropic radial nanopatterns are produced on polished titanium surfaces by spin-coating CNC at high speed. Furthermore, CNC surface chemistry allows to further sequester and form a coating of bioactive molecules derived from PL. The surface anisotropy provided by CNC guides fibroblasts growth and alignment up to 14 days of culture. Moreover, PL-derived biomolecules polarize macrophages toward the M2-like anti-inflammatory phenotype. These results suggest that the developed multifunctional surfaces can promote soft tissue integration to metallic implants and, at the same time, prevent bacterial invasion, tissue inflammation, and failure of biomedical metallic implants.

Magnetic Nanocomposite Hydrogels for Tissue Engineering: Design Concepts and Remote Actuation Strategies to Control Cell Fate.

Most tissues of the human body are characterized by highly anisotropic physical properties and biological organization. Hydrogels have been proposed as scaffolding materials to construct artificial tissues due to their water-rich composition, biocompatibility, and tunable properties. However, unmodified hydrogels are typically composed of randomly oriented polymer networks, resulting in homogeneous structures with isotropic properties different from those observed in biological systems. Magnetic materials have been proposed as potential agents to provide hydrogels with the anisotropy required for their use on tissue engineering. Moreover, the intrinsic properties of magnetic nanoparticles enable their use as magnetomechanic remote actuators to control the behavior of the cells encapsulated within the hydrogels under the application of external magnetic fields. In this review, we combine a detailed summary of the main strategies to prepare magnetic nanoparticles showing controlled properties with an analysis of the different approaches available to their incorporation into hydrogels. The application of magnetically responsive nanocomposite hydrogels in the engineering of different tissues is also reviewed.

Injectable hyaluronic acid and platelet lysate-derived granular hydrogels for biomedical applications.

Towards the repair of damaged tissues, numerous scaffolds have been fabricated to recreate the complex extracellular matrix (ECM) environment to support desired cell behaviors; however, it is often challenging to design scaffolds with the requisite cell-anchorage sites, mechanical stability, and tailorable physicochemical properties necessary for many applications. To address this and to improve on the properties of hyaluronic acid (HA) hydrogels, we combined photocrosslinkable norbornene-modified HA (NorHA) with human platelet lysate (PL). These PL-NorHA hybrid hydrogels supported the adhesion of cells when compared to NorHA hydrogels without PL, exhibited tailorable physicochemical properties based on the concentration of individual components, and released proteins over time. Using microfluidic techniques with on-chip mixing of NorHA and PL and subsequent photocrosslinking, spherical PL-NorHA microgels with a hierarchical fibrillar network were fabricated that exhibited the sustained delivery of PL proteins. Microgels could be jammed into granular hydrogels that exhibited shear-thinning and self-healing properties, enabling ejection from syringes and the fabrication of stable 3D constructs with 3D printing. Again, the inclusion of PL enhanced cellular interactions with the microgel structures. Overall, the combination of biomolecules and fibrin self-assembly arising from the enriched milieu of PL-derived proteins improved the bioactivity of HA-based hydrogels, enabling the formation of dynamic systems with modular design. The granular systems can be engineered to meet the complex demands of functional tissue repair using versatile processing techniques, such as with 3D printing.