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Natana Chaves Rabelo,1– 3 Maria Eduarda Gomes,1– 3 Isabelle de Oliveira Moraes,1– 3 Juliana Cantagalli Pfisterer,1– 3 Guilherme Loss de Morais,4 Deborah Antunes,5 Ernesto Raúl Caffarena,6 Juan Llerena Jr,1,2,7,8 Sayonara Gonzalez1– 3 1Centro de Genética Médica IFF/Fiocruz, Rio de Janeiro, RJ, Brazil; 2Centro de Referência para Doenças Raras IFF/Fiocruz, Rio de Janeiro, RJ, Brazil; 3Laboratório de Medicina Genômica IFF/Fiocruz, Rio de Janeiro, RJ, Brazil; 4Laboratório Nacional de Computação Científica, Petrópolis, RJ, Brazil; 5Laboratório de Genômica Funcional e Bioinformática, Instituto Oswaldo Cruz/Fiocruz, Rio de Janeiro, RJ, Brazil; 6Grupo de Biofísica Computacional e Modelagem Molecular, Programa de Computação Científica, Fundação Oswaldo Cruz/Fiocruz, Rio de Janeiro, RJ, Brazil; 7Faculdade de Medicina de Petrópolis, FASE, Petrópolis, RJ, Brazil; 8INAGEMP, Rio de Janeiro, RJ, BrazilCorrespondence: Juan Llerena Jr, Email juan.llerena@iff.fiocruz.brPurpose: Noonan syndrome and related disorders are genetic conditions affecting 1:1000– 2000 individuals. Variants causing hyperactivation of the RAS/MAPK pathway lead to phenotypic overlap between syndromes, in addition to an increased risk of pediatric tumors. DNA sequencing methods have been optimized to provide a molecular diagnosis for clinical and genetic heterogeneity conditions. This work aimed to investigate the genetic basis in RASopathy patients through Next Generation Sequencing in a Reference Center for Rare Diseases (IFF/Fiocruz) and implement the precision medicine at a public health institute in Brazil.Patients and Methods: This study comprises 26 cases with clinical suspicion of RASopathies. Sanger sequencing was used to screen variants in exons usually affected in the PTPN11 and HRAS genes for cases with clinical features of Noonan and Costello syndrome, respectively. Posteriorly, negative and new cases with clinical suspicion of RASopathy were analyzed by clinical or whole-exome sequencing.Results: Molecular analysis revealed recurrent variants and a novel LZTR1 missense variant: 24 unrelated individuals with pathogenic variants [PTPN11(11), NF1(2), SOS1(2), SHOC2(2), HRAS(1), BRAF(1), LZTR (1), RAF1(1), KRAS(1), RIT1(1), a patient with co-occurrence of PTPN11 and NF1 mutations (1)]; familial cases carrying a known pathogenic variant in PTPN11 (mother-two children), and a previously undescribed paternally inherited variant in LZTR1. The comparative modeling analysis of the novel LZTR1 variant p.Pro225Leu showed local and global changes in the secondary and tertiary structures, showing a decrease of about 1% in the β-sheet content. Furthermore, evolutionary conservation indicated that Pro225 is in a highly conserved region, as observed for known dominant pathogenic variants in this protein.Conclusion: Bringing precision medicine through NGS towards congenital syndromes promotes a better understanding of complex clinical and/or undiagnosed cases. The National Policy for Rare Diseases in Brazil emphasizes the importance of incorporating and optimizing diagnostic methodologies in the Unified Brazilian Health System (SUS). Therefore, this work is an important step for the NGS inclusion in diagnostic genetic routine in the public health system.Keywords: RASopathy, RAS/MAPK pathway, genetic heterogeneity, cancer risk, DNA sequencing

Abstract Background The absence of robust evidence of safety of medicines in pregnancy, particularly those for major diseases provided by public health programmes in developing countries, has resulted in cautious recommendations on their use. We describe a protocol for a Pregnancy Registry adapted to resource-limited settings aimed at providing evidence on the safety of medicines in pregnancy. Methods/Design Sentinel health facilities are chosen where women come for prenatal care and are likely to come for delivery. Staff capacity is improved to provide better care during the pregnancy, to identify visible birth defects at delivery and refer infants with major anomalies for surgical or clinical evaluation and treatment. Consenting women are enrolled at their first antenatal visit and careful medical, obstetric and drug-exposure histories taken; medical record linkage is encouraged. Enrolled women are followed up prospectively and their histories are updated at each subsequent visit. The enrolled woman is encouraged to deliver at the facility, where she and her baby can be assessed. Discussion In addition to data pooling into a common WHO database, the WHO Pregnancy Registry has three important features: First is the inclusion of pregnant women coming for antenatal care, enabling comparison of birth outcomes of women who have been exposed to a medicine with those who have not. Second is its applicability to resource-poor settings regardless of drug or disease. Third is improvement of reproductive health care during pregnancies and at delivery. Facility delivery enables better health outcomes, timely evaluation and management of the newborn, and the collection of reliable clinical data. The Registry aims to improves maternal and neonatal care and also provide much needed information on the safety of medicines in pregnancy.

Abstract A response to and comment on Pre-referral rectal artesunate in severe malaria: a flawed trial, by Karim F Hirji and Zulfiqarali G Premji.

Abstract Background Safety data regarding exposure to artemisinin-based combination therapy in pregnancy are limited. This prospective cohort study conducted in Zambia evaluated the safety of artemether-lumefantrine (AL) in pregnant women with malaria. Methods Pregnant women attending antenatal clinics were assigned to groups based on the drug used to treat their most recent malaria episode (AL vs. sulphadoxine-pyrimethamine, SP). Safety was assessed using standard and pregnancy-specific parameters. Post-delivery follow-up was six weeks for mothers and 12 months for live births. Primary outcome was perinatal mortality (stillbirth or neonatal death within seven days after birth). Results Data from 1,001 pregnant women (AL n = 495; SP n = 506) and 933 newborns (AL n = 466; SP n = 467) showed: perinatal mortality (AL 4.2%; SP 5.0%), comprised of early neonatal mortality (each group 2.3%), stillbirths (AL 1.9%; SP 2.7%); preterm deliveries (AL 14.1%; SP 17.4% of foetuses); and gestational age-adjusted low birth weight (AL 9.0%; SP 7.7%). Infant birth defect incidence was 1.8% AL and 1.6% SP, excluding umbilical hernia. Abortions prior to antenatal care could not be determined: abortion occurred in 4.5% of women treated with AL during their first trimester; none were reported in the 133 women exposed to SP and/or quinine during their first trimester. Overall development (including neurological assessment) was similar in both groups. Conclusions These data suggest that exposure to AL in pregnancy, including first trimester, is not associated with particular safety risks in terms of perinatal mortality, malformations, or developmental impairment. However, more data are required on AL use during the first trimester.

Abstract Background Malaria kills. A single rectal dose of artesunate before referral can reduce mortality and prevent permanent disability. However, the success of this intervention depends on caretakers' adherence to referral advice for follow-up care. This paper explores the dilemma facing caretakers when they are in the process of deciding whether or not to transit their child to a health facility after pre-referral treatment with rectal artesunate. Methods Four focus group discussions were held in each of three purposively selected villages in Mtwara rural district of Tanzania. Data were analysed manually using latent qualitative content analysis. Results The theme "Caretakers dilemma in deciding whether or not to adhere with referral advice after pre-referral treatment with rectal artesunate" depicts the challenge they face. Caretakers' understanding of the rationale for going to hospital after treatment - when and why they should adhere - influenced adherence. Caretakers, whose children did not improve, usually adhered to referral advice. If a child had noticeably improved with pre-referral treatment however, caretakers weighed whether they should proceed to the facility, balancing the child's improved condition against other competing priorities, difficulties in reaching the health facilities, and the perceived quality of care at the health facility. Some misinterpretation were found regarding the urgency and rationale for adherence among some caretakers of children who improved which were attributed to be possibly due to their prior understanding. Conclusion Some caretakers did not adhere when their children improved and some who adhered did so without understanding why they should proceed to the facility. Successful implementation of the rectal artesunate strategy depends upon effective communication regarding referral to clinic.

Abstract Background Rectal administration of artemisinin derivatives has potential for early treatment for severe malaria in remote settings where injectable antimalarial therapy may not be feasible. Preparations available include artesunate, artemisinin, artemether and dihydroartemisinin. However each may have different pharmacokinetic properties and more information is needed to determine optimal dose and comparative efficacy with each another and with conventional parenteral treatments for severe malaria. Methods Individual patient data from 1167 patients in 15 clinical trials of rectal artemisinin derivative therapy (artesunate, artemisinin and artemether) were pooled in order to compare the rapidity of clearance of Plasmodium falciparum parasitaemia and the incidence of reported adverse events with each treatment. Data from patients who received comparator treatment (parenteral artemisinin derivative or quinine) were also included. Primary endpoints included percentage reductions in parasitaemia at 12 and 24 hours. A parasite reduction of >90% at 24 hours was defined as parasitological success. Results Artemisinin and artesunate treatment cleared parasites more rapidly than parenteral quinine during the first 24 hours of treatment. A single higher dose of rectal artesunate treatment was five times more likely to achieve >90% parasite reductions at 24 hours than were multiple lower doses of rectal artesunate, or a single lower dose administration of rectal artemether. Conclusion Artemisinin and artesunate suppositories rapidly eliminate parasites and appear to be safe. There are less data on artemether and dihydroartemisinin suppositories. The more rapid parasite clearance of single high-dose regimens suggests that achieving immediate high drug concentrations may be the optimal strategy.

Abstract Introduction This paper reviews recent trends in the production, supply and price of the active ingredients as well as finished ACT products. Production and cost data provided in this paper are based on an ongoing project (Artepal). Stability data are derived from a development project on rectal artesunate. Discussion The artemisinin raw material and its derivatives appear to be very stable compared to the finished products. Supply of artemisinin changed in May 2004 when the Global Fund shifted financial support to qualified countries from chloroquine or sulphadoxine-pyrimethamine to an ACT for treatment of malaria. First, there was a sudden shortage of the starting material, and short term scarcity led to a steep rise in API price: it increased dramatically in 2004, from $350 per kg to more than $1000. Second, there was a parallel increase in the number of companies extracting artemisinin from 10 to 80 between 2003 and 2005 in China, and from 3 to 20 in Vietnam. Commercial cultivation began also in East Africa and Madagascar. A steady and predictable demand for the crop can eliminate such wide fluctuations and indirectly contribute to price stability of the herb, the API and ACT. With appropriate mechanisms to reduce those fluctuations, the cost of artemisinin might decrease sustainably to US$ 250–300 per kg. Conclusion Today the global health community is facing the risk of another cyclical swing with lower demand feeding into reduced planting of A. annua and, thereafter, a new shortage of the raw material and higher API prices. International donors, the largest purchasers for ACTs could better coordinate their activities, in order to guarantee purchase of ACTs and consequently of API with manufacturers. In parallel, the base of quality producers of APIs and finished ACT products needs to be broadened. While the ACT programme is still in its early stages, the consequences of another wave of artemisinin and ACT shortages would permanently discredit it and impede any progress in rolling malaria back.

Bacillus species are extensively documented as plant growth-promoting rhizobacteria, contributing significantly to the enhancement of soil fertility, nutrient recycling, and the control of phytopathogens. Utilizing them as biocontrol agents represents an environmentally friendly strategy, particularly within the rhizospheric community. This study presents the comprehensive genome sequences of three B. velezensis strains (LGMB12, LGMB319, and LGMB426) which were previously isolated from root samples of maize (Zea mays L.), along with a type strain FZB42. The research assesses the capability of the three strains for antagonizing fungi, specifically Fusarium graminearum, Fusarium verticillioides, Colletotrichum graminicola, and Stenocarpella sp. In paired cultures involving maize fungi, treatments containing bacteria B. velezensis exhibited statistically significant differences compared to both negative and positive treatments in terms of antagonism. Furthermore, genome mining techniques were employed to explore their inherent antagonistic potential. The assembly revealed that strains LGMB12, LGMB319, LGMB426, and FZB42 exhibit genome sizes of 4,187,541 bp, 4,244,954 bp, 3,976,537 bp, and 3,990,518 respectively. Their respective G + C content stands at 46.42 %, 46.50 %, 46.51 %, and 46.38 %. Moreover, the genomes present multiple gene clusters responsible for the synthesis of secondary metabolites and carbohydrate-active enzymes (CAZymes). These clusters highlight a diverse array of antibacterial and antifungal properties, complemented by numerous plant growth-promoting genes. These results highlight the potential of B. velezensis LGMB12, LGMB319, and LGMB426 strains as biocontrol and plant growth promotion agents, being promising candidates for further studies in agricultural production, including field trials., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

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., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

There is growing evidence on the significant role of prolonged inflammation in triggering and progressing of numerous diseases with substantial health and socioeconomic impacts, such as musculoskeletal, cardiovascular and autoimmune disorders, and cancer. Therefore, there is an urgent need to develop therapies that can overcome the main challenges of currently used approaches, such as non-target action, partial modulation of the complex inflammatory pathways, and short-term effects, to effectively manage and resolve chronic inflammatory states. This work investigates the therapeutic synergy of clinically relevant anti-inflammatory agents approaching naïve and classically activated macrophages owing to their central role in inflammation. Aiming at human therapies, a dual-loading nanoplatform reunites molecules with different physico-chemical properties in a single system, seeking to more effectively and comprehensively regulate macrophage functions for precision cell guidance and greater versatility in disease managing. To build this platform, palmitic acid grafted chitosan, superparamagnetic iron oxide nanoparticles, the clinically approved NSAID celecoxib (also known as Celebrex ® ), and RNA technologies were combined into superparamagnetic polymeric micelles (SPMs). Our findings demonstrated that traditional anti-inflammatory drugs such as celecoxib and microRNA molecules were efficiently delivered by the SPMs, altering the inflammatory profile of naïve (M0φ) and M1-primed macrophages (M1φ) assessed by gene and protein expression. The impact of the dual-loaded SPMs in naïve Mφ is an interesting finding towards the modulation of the initial immune response, reducing the potential for chronic inflammation and promoting tissue healing. Collectively, these encouraging results demonstrate the promise of multi-nanomedicine strategies to enhance the efficacy of therapeutic interventions by offering a fresh approach to more precisely and carefully regulated nanotherapeutics delivery.

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-1β-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-1β-primed-magCSs with an involvement of the NF-κB signaling pathway. Encouragingly, when PEMF was applied to IL-1β-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.

Cartilage-hair hypoplasia syndrome (CHH) is an autosomal recessive disorder frequently linked to n.72A>G (previously known as n.70A>G and n.71A>G), the most common RMRP variant worldwide. More than 130 pathogenic variants in this gene have already been described associated with CHH, and founder alterations were reported in the Finnish and Japanese populations. Our previous study in Brazilian CHH patients showed a high prevalence of n.197C>T variant (former n.195C>T and n.196C>T) when compared to other populations. The aim of this study was to investigate a possible founder effect of the n.197C>T variant in the RMRP gene in a series of CHH Brazilian patients. We have selected four TAG SNPs within chromosome 9 and genotyped the probands and their parents (23 patients previously described and nine novel). A common haplotype to the n.197C>T variant carriers was identified. Patients were also characterized for 46 autosomal Ancestry Informative Markers (AIMs). European ancestry was the most prevalent (58%), followed by African (24%) and Native American (18%). Our results strengthen the hypothesis of a founder effect for the n.197C>T variant in Brazil and indicate that this variant in the RMRP gene originated from a single event on chromosome 9 with a possible European origin., (© 2024. The Author(s).)

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., (© 2024 The Authors. Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

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.

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.

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.

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.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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.

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.

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.

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., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

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.

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.

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.

Anthracnose, caused by fungi of the genus Colletotrichum, is present in the main areas where rubber trees ( Hevea brasiliensis ) are planted. Thus, considering that biological agents can be an alternative for disease control, the present study aimed to carry out initial studies to investigate the response of rubber tree seedlings inoculated with Colletotrichum and treated with saprobes fungi from the semiarid region of Northeast Brazil ( Curvularia eragrostidis , Memnoniella levispora , Myrothecium roridum and Phialomyces macrosporus ). Seedlings of the rubber tree clone RRIM600 were sprayed with biocontrol agents as preventive and curative treatments seven days before and after C. tamarilloi inoculation, respectively. Assessments included plant response to disease expression based on the percentage of symptomatic area on treated leaves, percentage of graft death, and percentage of apical death in seedlings 30 days after inoculation with C. tamarilloi . In addition, the enzymes peroxidase and phenylalanine ammonia lyase (PAL) had their activity quantified by their association with plant resistance to pathogens. The fungus C. eragrostidis had the best result in controlling anthracnose when applied as a preventive treatment, showing 10% less disease than the untreated plant. The same was observed for the fungus P. macrosporus when used in the curative form. These fungi also reduced the graft death. In these cases, PAL activity was higher and may be linked to the induction of resistance against the pathogen. The peroxidase activity was not expressive for treatments with saprobic fungi in the periods studied. Therefore, among the tested fungi, C. eragrostidis and P. macrosporus are promising for the control of anthracnose, deserving further studies.

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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Bioengineered human skeletal muscle tissues have emerged in the last years as new in vitro systems 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., (Creative Commons Attribution license.)

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., Competing Interests: S.R. was an early career researcher recruited at Sofradim Production, Medtronic, France and registered for PhD at NUI Galway, Ireland. Y.B. is an employee of Sofradim Production, Medtronic, France. E.P., S.H.K., E.M.F., M.E.G., R.L.R., A.O'R, and D.I.Z. declare no conflicts of interest., (© 2022 The Authors. Engineering in Life Sciences published by Wiley‐VCH GmbH.)

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., (© 2022 Wiley-VCH GmbH.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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.

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., (© 2022 Wiley-VCH GmbH.)

Anthracnose, caused by fungi of the genus Colletotrichum , is present in the major rubber tree crop areas in Brazil, especially in São Paulo, Mato Grosso do Sul, Paraná, Minas Gerais, Espírito Santo, and northern states. This disease can affect different tissues of the rubber tree, leading to production losses. Thus, a better understanding of the pathosystem Colletotrichum x rubber tree can provide evidence to subsequent epidemiological research and phytosanitary management studies of this disease in the field. The present study aimed to investigate C. tamarilloi colonization and reproduction steps in resistant clones (IAC 502, IAC 507, RRIM 937) and in one susceptible clone (RRIM 600) of the rubber tree, verifying the influence of temperature up to 48 h after inoculation of the fungus, under 24 h wetness. Samples were analyzed under a light, a UV and a scanning electron microscope. Data indicated that the fungus had a delay in its development in resistant clones and, although colonization was expressive 48 h after inoculation, the new spore formation rate in the analyzed samples was lower in resistant clones. For RRIM 600, rapid colonization and intensive sporulation could be observed.

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.

Recurrent microbial infections are a major cause of surgical failure and morbidity. Wound healing strategies based on hydrogels have been proposed to provide at once a barrier against pathogen microbial colonization, as well as a favorable environment for tissue repair. Nevertheless, most biocompatible hydrogel materials are more bacteriostatic than antimicrobial materials, and lack specific action against pathogens. Silver-loaded polymeric nanocomposites have efficient and selective activity against pathogenic organisms exploitable for wound healing. However, the loading of metallic nanostructures into hydrogels represents a major challenge due to the low stability of metal colloids in aqueous environments. In this context, the aim of the present study was the development of highly stable silver nanoparticles (AgNPs) as novel potential antimicrobial agents for hyaluronic acids hydrogels. Two candidate stabilizing agents obtained from natural and renewable sources, namely cellulose nanocrystals and ulvan polysaccharide, were exploited to ensure high stability of the silver colloid. Both stabilizing agents possess inherent bioactivity and biocompatibility, as well as the ability to stabilize metal nanostructures thanks to their supramolecular structures. Silver nitrate reduction through sodium borohydride in presence of the selected stabilizing agents was adopted as a model strategy to achieve AgNPs with narrow size distribution. Optimized AgNPs stabilized with the two investigated polysaccharides demonstrated high stability in phosphate buffer saline solution and strong antimicrobial activity. Loading of the developed AgNPs into photocrosslinked methacrylated hyaluronic acid hydrogels was also investigated for the first time as an effective strategy to develop novel antimicrobial wound dressing materials.

Tendon and ligament (T/L) engineering strategies towards clinical practice have been challenged by a paucity of understanding in the identification and still poorly described characterization of cellular niches. Prospecting how resident cell populations behave in vitro, and how cryopreservation may influence T/ L-promoting factors, can provide insights into T/ L-cellular profiles for novel regenerative solutions. Therefore, we studied human T/ L-derived cells isolated from patellar tendons and cruciate ligaments as suitable cellular models to anticipate tendon and ligament niches responses for advanced strategies with predictive tenogenic and ligamentogenic value. Our results show that the crude populations isolated from tendon and ligament tissues hold a stem cell subset and share a similar behavior in terms of tenogenic/ligamentogenic commitment. Both T/ L-derived cells successfully undergo cryopreservation/thawing maintaining the tenogenic/ligamentogenic profiles. The major differences between cryopreserved and fresh populations were observed at the gene expression of MKX, SCX, and TNMD as well as at the protein levels of collagen type I and III, in which cells from tendon origin (hTDCs) evidence increased values in comparison to the ones from ligament (hLDCs, p < 0.05). In addition, low-temperature storage was shown to potentiate an immunomodulatory profile of cells, especially in hTDCs leading to an increase in the gene expression of the anti-inflammatory factors IL-4 and IL-10 (p < 0.05), as well as in the protein secretion of IL-10 (p < 0.01) and IL-4 (p < 0.001). Overall, the outcomes highlight the relevance of the cryopreserved T/ L-derived cells and their promising immunomodulatory cues as in vitro models for investigating cell-mediated mechanisms driving tissue healing and regeneration., (© 2021 Wiley Periodicals LLC.)

Dental pulp tissue engineering (TE) endeavors to regenerate dentin/pulp complex by combining a suitable supporting matrix, stem cells, and biochemical stimuli. Such procedures foresee a matrix that can be easily introduced into the root canal system (RCS) and tightly adhere to dentin walls to assure the dentin surface's proper colonization with progenitor cells capable of restoring the dentin/pulp complex. Herein was investigated an injectable self-setting hyaluronic acid-based (HA) hydrogel system, formed by aldehyde-modified (a-HA) with hydrazide-modified (ADH), enriched with platelet lysate (PL), for endodontic regeneration. The hydrogels' working (wT) and setting (sT) times, the adhesion to the dentine walls, the hydrogel's microstructure, and the delivery of human dental pulp cells (DPCs) were studied in vitro. Hydrogels incorporating PL showed a suitable wT and sT and a porous microstructure. The tensile tests showed that the breaking point occurs after 4.3106 ± 1.8677 mm deformation, while in the indentation test after 1.4056 ± 0.3065 mm deformation. Both breaking points occur in the hydrogel extension. The HA/PL hydrogels exhibited supportive properties and promoted cell migration toward dentin surfaces in vitro. Overall, these results support using PL-laden HA injectable hydrogels (HA/PL) as a biomaterial for DPCs encapsulation, thereby displaying great clinical potential towards endodontic regenerative therapies.

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.

Cell sheet technology and magnetic based tissue engineering hold the potential to become instrumental in developing magnetically responsive living tissues analogues that can be potentially used both for modeling and therapeutical purposes. Cell sheet constructions more closely recreate physiological niches, through the preservation of contiguous cells and cell-ECM interactions, which assist the cellular guidance in regenerative processes. We herein propose to use magnetically assisted cell sheets (magCSs) constructed with human tendon-derived cells (hTDCs) and magnetic nanoparticles to study inflammation activity upon magCSs exposure to IL-1β, anticipating its added value for tendon disease modeling. Our results show that IL-1β induces an inflammatory profile in magCSs, supporting its in vitro use to enlighten inflammation mediated events in tendon cells. Moreover, the response of magCSs to IL-1β is modulated by pulsed electromagnetic field (PEMF) stimulation, favoring the expression of anti-inflammatory genes, which seems to be associated to MAPK(ERK1/2) pathway. The anti-inflammatory response to PEMF together with the immunomodulatory potential of magCSs opens new perspectives for their applicability on tendon regeneration that goes beyond advanced cell based modeling. STATEMENT OF SIGNIFICANCE: The combination of cell sheets and magnetic-based technologies holds promise as instrumental bio-instructive tools both for tendon disease modelling and for the development of magnetically responsive living tendon substitutes. We have previously shown that remote actuation of a pulsed electromagnetic field (PEMF) modulated the inflammatory response of IL-1β-treated human tendon-derived cell (hTDCs) monolayers. As magnetic cell sheets (magCSs) technologies enable improved cellular organization and matrix deposition, these constructions could better recapitulate tendon niches. In this work, we aimed to apply magCSs technologies to study hTDCs responses in inflammatory environments. Overall results show that PEMF-stimulated-magCSs hold evidence for immunomodulatory properties and to become a living tendon model envisioning tendon regenerative therapies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)