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The lack of representative in vitro models recapitulating human tendon (patho)physiology is among the major factors hindering consistent progress in the knowledge-based development of adequate therapies for tendinopathy.Here, an organotypic 3D tendon-on-chip model is designed that allows studying the spatiotemporal dynamics of its cellular and molecular mechanisms.Combining the synergistic effects of a bioactive hydrogel matrix with the biophysical cues of magnetic microfibers directly aligned on the microfluidic chip, it is possible to recreate the anisotropic architecture, cell patterns, and phenotype of tendon intrinsic (core) compartment. When incorporated with vascular-like vessels emulating the interface between its intrinsic-extrinsic compartments, crosstalk with endothelial cells are found to drive stromal tenocytes toward a reparative profile. This platform is further used to study adaptive immune cell responses at the onset of tissue inflammation, focusing on interactions between tendon compartment tenocytes and circulating T cells.The proinflammatory signature resulting from this intra/inter-cellular communication induces the recruitment of T cells into the inflamed core compartment and confirms the involvement of this cellular crosstalk in positive feedback loops leading to the amplification of tendon inflammation.Overall, the developed 3D tendon-on-chip provides a powerful new tool enabling mechanistic studies on the pathogenesis of tendinopathy as well as for assessing new therapies.

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Tissue environments play a crucial role in orchestrating cell behavior, guided by a complex interplay of various factors. Long lasting inflammatory signals compromise tendon homeostasis and promote tissue degeneration, while tissue oxygen levels affect local cells' responses with hypoxic environments influencing apoptosis, inflammatory mediators, and matrix production. Recent works have unveiled the therapeutic potential of pulsed electromagnetic field (PEMF) in modulating inflammatory signals expressed by human tendon cells (hTDCs), and in mitigating the hypoxia-induced effects on the regulation of inflammatory cytokines. Thus, we sought to investigate the role of hypoxic environments, namely, 1 and 2% oxygen tension, in the inflammatory profiles of magnetic cell sheets (magCSs) formed by magnetic nanoparticles internalized in contiguous hTDCs with intact cell-cell junctions and deposited matrix. We also aimed to explore the impact of PEMF over hypoxia-treated magCSs, including IL-1ß-primed-magCSs, with the objective of harnessing magnetic stimulation to guide abnormal inflammatory cell responses toward efficient treatments supporting tendon regenerative potential. Our findings revealed that low oxygen tensions amplified the expression of hypoxia-associated genes and of inflammatory markers in IL-1ß-primed-magCSs with an involvement of the NF-κB signaling pathway. Encouragingly, when PEMF was applied to IL-1ß-primed-magCSs under hypoxic conditions, it successfully modulated inflammatory cues by favoring IL-10 and IL-4, via the NF-κB pathway. These results signify the remarkable potential of PEMF in driving proregenerative strategies and opens up new approaches in tendon therapies, highlighting the transformative impact of immunomodulatory magnetic cell sheets.

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Background: Low fibrinogen levels are associated with an increased risk of perioperative bleeding. However, there is an ongoing debate over the ideal treatment threshold, the benefits of prophylactic supplementation with fibrinogen concentrate, and the best source of fibrinogen. While fibrinogen concentrate supplementation is being widely used to treat bleeding related to acquired haemostatic deficiencies, there is a lack of evidence regarding its dosage, effectiveness, and safety. This systematic review provides an up-to-date summary of the relationship between fibrinogen concentrate supplementation and safety measures in the perioperative care of non-trauma, non-obstetric adult patients. Methods: A comprehensive online search was conducted on PubMed/Medline, EMBASE, Scopus, Web of Science, the Cochrane Database of Systematic Reviews, and the Cochrane Central Register of Controlled Trials. Results: This systematic review and meta-analysis encompasses ten studies involving 1391 patients. There was a decreased risk of total thromboembolic events in patients treated with fibrinogen compared to the control (OR 0.65, 95% CI 0.43 to 0.98, I2 = 0%). In addition, when fibrinogen was used prophylactically, it resulted in shorter ICU stays (MD -1.50, 95% CI -2.64 to -0.36), when set against its therapeutic use. A sensitivity analysis on cardiovascular surgery studies did not reveal any statistically significant difference. Conclusions: The use of fibrinogen concentrate in the perioperative care of non-trauma and non-obstetric adult patients may lead to potential benefits.

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[This corrects the article DOI: 10.1371/journal.pone.0083734.].

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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.

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Three-dimensional (3D) printing technology has become a popular tool to produce complex structures. It has great potential in the regenerative medicine field to produce customizable and reproducible scaffolds with high control of dimensions and porosity. This study was focused on the investigation of new biocompatible and biodegradable 3D-printed scaffolds with suitable mechanical properties to assist tendon and ligament regeneration. Polylactic acid (PLA) scaffolds were reinforced with 0.5 wt.% of functionalized graphite nanoplatelets decorated with silver nanoparticles ((f-EG)+Ag). The functionalization of graphene was carried out to strengthen the interface with the polymer. (f-EG)+Ag exhibited antibacterial properties against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli), an important feature for the healing process and prevention of bacterial infections. The scaffolds' structure, biodegradation, and mechanical properties were assessed to confirm their suitability for tendon and ligamentregeneration. All scaffolds exhibited surface nanoroughness created during printing, which was increased by the filler presence. The wet state dynamic mechanical analysis proved that the incorporation of reinforcement led to an increase in the storage modulus, compared with neat PLA. The cytotoxicity assays using L929 fibroblasts showed that the scaffolds were biocompatible. The PLA+[(f-EG)+Ag] scaffolds were also loaded with human tendon-derived cells and showed their capability to maintain the tenogenic commitment with an increase in the gene expression of specific tendon/ligament-related markers. The results demonstrate the potential application of these new 3D-printed nanocomposite scaffolds for tendon and ligament regeneration.

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In recent years, nanotechnology-based microRNA (miR) therapeutic platforms have shown great promise for immunotherapy and tissue regeneration, despite the unmet challenge of achieving efficient and safe delivery of miRs. The transport of miRs offers precision and regulatory value for a myriad of biological processes and pathways, including the control of macrophage (Mφ) functions and, consequently, the inflammatory cascades Mφ are involved in. Thus, enforcement of Mφ can boost the regenerative process and provide new solutions for diverse chronic pathologies. In this study, we sought to develop a magnetically guided transporter to deliver an miR-155 antagonist to M1-primed Mφ. Furthermore, we determined its modulatory effect in reprogramming Mφ from inflammatory to pro-regenerative phenotypes, with the aim of tissue healing and regenerative medicine approaches. This strategy combines contactless and high-precision control of Mφ, anticipating new functional miR carriers for targeted strategies controlled by extracorporeal action. The magnetoplexes SPION@PEI-miR were efficiently delivered into Mφ without compromising cell viability and successfully induced miR-mediated gene silencing by enhancing the expression of anti-inflammatory markers (IL4 and IL10) and the production of M2φ-related markers (CD206 and IL4). Given its multimodal features, SPION@PEI-miR represents a simple, safe, and nonviral theranostic platform that enables imaging, tracking, and miR delivery with modulatory effects on immune cells.

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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.

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Tendons are mechanosensitive connective tissues responsible for the connection between muscles and bones by transmitting forces that allow the movement of the body, yet, with advancing age, tendons become more prone to degeneration followed by injuries. Tendon diseases are one of the main causes of incapacity worldwide, leading to changes in tendon composition, structure, and biomechanical properties, as well as a decline in regenerative potential. There is still a great lack of knowledge regarding tendon cellular and molecular biology, interplay between biochemistry and biomechanics, and the complex pathomechanisms involved in tendon diseases. Consequently, this reflects a huge need for basic and clinical research to better elucidate the nature of healthy tendon tissue and also tendon aging process and associated diseases. This chapter concisely describes the effects that the aging process has on tendons at the tissue, cellular, and molecular levels and briefly reviews potential biological predictors of tendon aging. Recent research findings that are herein reviewed and discussed might contribute to the development of precision tendon therapies targeting the elderly population.

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The paradigm shift in the endodontic field from replacement toward regenerative therapies has witnessed the ever-growing research in tissue engineering and regenerative medicine targeting pulp-dentin complex in the past few years. Abundant literature on the subject that has been produced, however, is scattered over diverse areas of knowledge. Moreover, the terminology and concepts are not always consensual, reflecting the range of research fields addressing this subject, from endodontics to biology, genetics, and engineering, among others. This fact triggered some misinterpretations, mainly when the denominations of different approaches were used as synonyms. The evaluation of results is not precise, leading to biased conjectures. Therefore, this literature review aims to conceptualize the commonly used terminology, summarize the main research areas on pulp regeneration, identify future trends, and ultimately clarify whether we are really on the edge of a paradigm shift in contemporary endodontics toward pulp regeneration.

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Tendon afflictions constitute a significant share of musculoskeletal diseases and represent a primary cause of incapacity worldwide. Unresolved/chronic inflammatory states have been associated with the onset and progression of tendon disorders, contributing to undesirable immune stimulation and detrimental tissue effects. Thus, targeting persistent inflammatory events could assist important developments to solve pathophysiological processes and innovative therapeutics to address impaired healing and accomplish complete tendon regeneration. This review overviews the impact of inflammation and inflammatory mediators in tendon niches, unveiling the importance of tendon cell populations and their signature features, and the influence of microenvironmental factors on inflamed and injured tendons. The demand for non-invasive instructive strategies to manage persistent inflammatory mediators, guide inflammatory pathways, and modulate cellular responses will also be approached by exploring the role of pulsed electromagnetic field (PEMF). PEMF alone or combined with more sophisticated systems triggered by magnetic fields will be considered in the design of successful therapies to control inflammation in tendinopathic conditions.

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The tooth is made up of three mineralized tissues, enamel, dentin, and cementum, which surround a non-mineralized tissue called the dental pulp. Micro-computed tomography (mCT) is an imaging technology based on X-rays that allows non-invasive visualization of objects at a microscopic scale, according to their radiopacity and in three dimensions (3D). Likewise, it allows the subsequent execution of morphological and quantitative analysis of the objects, such as, for example, the determination of the relative mineral density (MD). The present work aimed to describe the MD of feline teeth using mCT. The studied sample consisted of four European Shorthair cats, from which nine canine teeth were extracted per medical indication. These teeth were evaluated through dental radiography before and after their extraction. Using mCT and the CTAn software, the values of the relative mineral density of the root of each tooth and of specific segments corresponding to the coronal, middle, and apical thirds of the root were determined. Mean MD of root tissues was 1.374 ± 0040 g·cm-3, and of hard root, tissues was 1.402 ± 0.035 g·cm-3. Through mCT, it was possible to determine the mean MD values of feline canine teeth. The study of MD could become an ancillary method for the diagnosis and characterization of dental pathology.

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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.

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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.

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The persistence of inflammatory mediators in tissue niches significantly impacts regenerative outcomes and contributes to chronic diseases. Interleukin-4 (IL4) boosts pro-healing phenotypes in macrophages (Mφ) and triggers the activation of signal transducer and activator of transcription 6 (STAT6). Since the IL4/STAT6 pathway reduces Mφ responsiveness to inflammation in a targeted and precise manner, IL4 delivery offers personalized possibilities to overcome inflammatory events. Despite its therapeutic potential, the limited success of IL4-targeted delivery is hampered by inefficient vehicles. Magnetically assisted technologies offer precise and tunable nanodevices for the delivery of cytokines by combining contactless modulation, high tissue penetration, imaging features, and low interference with the biological environment. Although superparamagnetic iron oxide nanoparticles (SPION) have shown clinical applicability in imaging, SPION-based approaches have rarely been explored for targeted delivery and cell programming. Herein, we hypothesized that SPION-based carriers assist in efficient IL4 delivery to Mφ, favoring a pro-regenerative phenotype (M2φ). Our results confirmed the efficiency of SPION-IL4 and Mφ responsiveness to SPION-IL4 with evidence of STAT6-mediated polarization. SPION-IL4-treated Mφ showed increased expression of M2φ associated-mediators (IL10, ARG1, CCL2, IL1Ra) when compared to the well-established soluble IL4. The ability of SPION-IL4 to direct Mφ polarization using sophisticated magnetic nanotools is valuable for resolving inflammation and assisting innovative strategies for chronic inflammatory conditions.

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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.

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Hybrid nanoarchitectures such as magnetic polymeric micelles (MPMs) are among the most promising nanotechnology-enabled materials for biomedical applications combining the benefits of polymeric micelles and magnetic nanoparticles within a single bioinstructive system. MPMs are formed by the self-assembly of polymer amphiphiles above the critical micelle concentration, generating a colloidal structure with a hydrophobic core and a hydrophilic shell incorporating magnetic particles (MNPs) in one of the segments. MPMs have been investigated most prominently as contrast agents for magnetic resonance imaging (MRI), as heat generators in hyperthermia treatments, and as magnetic-susceptible nanocarriers for the delivery and release of therapeutic agents. The versatility of MPMs constitutes a powerful route to ultrasensitive, precise, and multifunctional diagnostic and therapeutic vehicles for the treatment of a wide range of pathologies. Although MPMs have been significantly explored for MRI and cancer therapy, MPMs are multipurpose functional units, widening their applicability into less expected fields of research such as bioengineering and regenerative medicine. Herein, we aim to review published reports of the last five years about MPMs concerning their structure and fabrication methods as well as their current and foreseen expectations for advanced biomedical applications.

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The combined effect of surface topography and substrate rigidity in stem cell cultures is still under-investigated, especially when biodegradable polymers are used. Herein, we assessed human bone marrow stem cell response on aliphatic polyester substrates as a function of anisotropic grooved topography and rigidity (7 and 12 kPa). Planar tissue culture plastic (TCP, 3 GPa) and aliphatic polyester substrates were used as controls. Cell morphology analysis revealed that grooved substrates caused nuclei orientation/alignment in the direction of the grooves. After 21 days in osteogenic and chondrogenic media, the 3 GPa TCP and the grooved 12 kPa substrate induced significantly higher calcium deposition and alkaline phosphatase (ALP) activity and glycosaminoglycan (GAG) deposition, respectively, than the other groups. After 14 days in tenogenic media, the 3 GPa TCP upregulated four and downregulated four genes; the planar 7 kPa substrate upregulated seven genes and downregulated one gene; and the grooved 12 kPa substrate upregulated seven genes and downregulated one gene. After 21 days in adipogenic media, the softest (7 kPa) substrates induced significantly higher oil droplet deposition than the other substrates and the grooved substrate induced significantly higher droplet deposition than the planar. Our data pave the way for more rational design of bioinspired constructs.