The Potential of the Fibronectin Inhibitor Arg-Gly-Asp-Ser in the Development of Therapies for Glioblastoma.

Glioblastoma (GBM) is the most lethal and common malignant primary brain tumor in adults. An important feature that supports GBM aggressiveness is the unique composition of its extracellular matrix (ECM). Particularly, fibronectin plays an important role in cancer cell adhesion, differentiation, proliferation, and chemoresistance. Thus, herein, a hydrogel with mechanical properties compatible with the brain and the ability to disrupt the dynamic and reciprocal interaction between fibronectin and tumor cells was produced. High-molecular-weight hyaluronic acid (HMW-HA) functionalized with the inhibitory fibronectin peptide Arg-Gly-Asp-Ser (RGDS) was used to produce the polymeric matrix. Liposomes encapsulating doxorubicin (DOX) were also included in the hydrogel to kill GBM cells. The resulting hydrogel containing liposomes with therapeutic DOX concentrations presented rheological properties like a healthy brain. In vitro assays demonstrated that unmodified HMW-HA hydrogels only caused GBM cell killing after DOX incorporation. Conversely, RGDS-functionalized hydrogels displayed per se cytotoxicity. As GBM cells produce several proteolytic enzymes capable of disrupting the peptide-HA bond, we selected MMP-2 to illustrate this phenomenon. Therefore, RGDS internalization can induce GBM cell apoptosis. Importantly, RGDS-functionalized hydrogel incorporating DOX efficiently damaged GBM cells without affecting astrocyte viability, proving its safety. Overall, the results demonstrate the potential of the RGDS-functionalized hydrogel to develop safe and effective GBM treatments.

Decellularized kidney extracellular matrix-based hydrogels for renal tissue engineering.

Kidney regeneration is hindered by the limited pool of intrinsic reparative cells. Advanced therapies targeting renal regeneration have the potential to alleviate the clinical and financial burdens associated with kidney disease. Delivery systems for cells, extracellular vesicles, or growth factors aimed at enhancing regeneration can benefit from vehicles enabling targeted delivery and controlled release. Hydrogels, optimized to carry biological cargo while promoting regeneration, have emerged as promising candidates for this purpose. This study aims to develop a hydrogel from decellularized kidney extracellular matrix (DKECM) and explore its biocompatibility as a biomaterial for renal regeneration. The resulting hydrogel crosslinks with temperature and exhibits a high concentration of extracellular matrix. The decellularization process efficiently removes detergent residues, yielding a pathogen-free biomaterial that is non-hemolytic and devoid of α-gal epitope. Upon interaction with macrophages, the hydrogel induces differentiation into both pro-inflammatory and anti-inflammatory phenotypes, suggesting an adequate balance to promote biomaterial functionality in vivo. Renal progenitor cells encapsulated in the DKECM hydrogel demonstrate higher viability and proliferation than in commercial collagen-I hydrogels, while also expressing tubular cells and podocyte markers in long-term culture. Overall, the injectable biomaterial derived from porcine DKECM is anticipated to elicit minimal host reaction while fostering progenitor cell bioactivity, offering a potential avenue for enhancing renal regeneration in clinical settings. STATEMENT OF SIGNIFICANCE: The quest to improve treatments for kidney disease is crucial, given the challenges faced by patients on dialysis or waiting for transplants. Exciting new therapies combining biomaterials with cells can revolutionize kidney repair. In this study, researchers created a hydrogel from pig kidney. This gel could be used to deliver cells and other substances that help in kidney regeneration. Despite coming from pigs, it's safe for use in humans, with no harmful substances and reduced risk of immune reactions. Importantly, it promotes a balanced healing response in the body. This research not only advances our knowledge of kidney repair but also offers hope for more effective treatments for kidney diseases.

COX-2 inhibitor delivery system aiming intestinal inflammatory disorders.

Selective COX-2 inhibitors such as etoricoxib (ETX) are potentially indicated for the treatment of intestinal inflammatory disorders. However, their systemic administration provokes some off-site secondary effects, decreasing the desirable local effectiveness. To circumvent such limitations, herein an ETX delivery system based on electrospun fibrous meshes (eFMs) was proposed. ETX at different concentrations (1, 2, and 3 mg mL-1) was loaded into eFMs, which not affect the morphology and the mechanical properties of this drug delivery system (DDS). The ETX showed a burst release within the first 12 h, followed by a faster release until 36 h, gradually decreasing over time. Importantly, the ETX studied concentrations were not toxic to human colonic cells (i.e. epithelial and fibroblast). Moreover, the DDS loading the highest concentration of ETX, when tested with stimulated human macrophages, promoted a reduction of PGE2, IL-8 and TNF-α secretion. Therefore, the proposed DDS may constitute a safe and efficient treatment of colorectal diseases promoted by inflammatory disorders associated with COX-2.

Diaphragmatic hernia repair porcine model to compare the performance of biodegradable membranes against Gore-Tex®.

BACKGROUND: Patch repair of congenital diaphragmatic hernia (CDH) using Gore-Tex® is associated with infection, adhesions, hernia recurrence, long-term musculoskeletal sequels and poor tissue regeneration. To overcome these limitations, the performance of two novel biodegradable membranes was tested to repair CDH in a growing pig model. METHODS: Twelve male pigs were randomly assigned to 3 different groups of 4 animals each, determined by the type of patch used during thoracoscopic diaphragmatic hernia repair (Gore-Tex®, polycaprolactone electrospun membrane-PCLem, and decellularized human chorion membrane-dHCM). After 7 weeks, all animals were euthanized, followed by necropsy for diaphragmatic evaluation and histological analysis. RESULTS: Thoracoscopic defect creation and diaphragmatic repair were performed without any technical difficulty in all groups. However, hernia recurrence rate was 0% in Gore-Tex®, 50% in PCLem and 100% in dHCM groups. At euthanasia, Gore-Tex® patches appeared virtually unchanged and covered with a fibrotic capsule, while PCLem and dHCM patches were replaced by either floppy connective tissue or vascularized and floppy regenerated membranous tissue, respectively. CONCLUSION: Gore-Tex® was associated with a higher survival rate and lower recurrence. Nevertheless, the proposed biodegradable membranes were associated with better tissue integration when compared with Gore-Tex®.

Primary Squamous Cell Carcinoma of the Renal Pelvis: A Rare Complication of Xanthogranulomatous Pyelonephritis.

Xanthogranulomatous pyelonephritis is a rare disease resulting from chronic inflammation and infection of the renal parenchyma. It usually arises as a consequence of obstructive chronic pyelonephritis. Primary squamous cell carcinoma of the renal pelvis is a distinct pathology, very rare in clinical practice, with a well-established association with xanthogranulomatous pyelonephritis. The authors present the case of a 57-year-old woman with chronic pyelonephritis containing xanthogranulomatous features. Subsequent workup revealed a concomitant, unsuspected, primary squamous cell carcinoma of the renal pelvis. With this case, the authors intend to emphasize and reinforce the need to be alert to an uncommon association between two rare diseases due to its diagnostic, therapeutic, and prognostic implications.

Supracondylar Apophysis of the Humerus: Rare Cause of High Compression of the Median Nerve.

Supracondylar apophysis (SA) is a bony prominence that originates from the anteromedial aspect of the distal humerus with a lower projection and which, although usually asymptomatic, due to the relationship with adjacent structures can cause symptoms. We describe the case of a 42-year-old woman with pain complaints radiating from her elbow to her hand, with 6 months of evolution. On objective examination, the patient had a sensory deficit in the median nerve territory and decreased grip strength. Radiographs of the distal humerus were performed, in which a bone spike was visible, and magnetic resonance imaging showed thickening of the median nerve epineurium. Electromyography showed severe axonal demyelination of the median nerve proximal to the elbow. A median nerve compression caused by a SA was diagnosed. The patient underwent surgery and, 1 year after the operation, she had a complete clinical recovery. Supracondylar apophysis is a rare, but possible and treatable cause of high median nerve compression.

Fast and Simple UPLC-Q-TOF MS Method for Determination of Bitter Flavan-3-ols and Oligomeric Proanthocyanidins: Impact of Vegetable Protein Fining Agents on Red Wine Composition.

Wine phenolic compounds, particularly proanthocyanidins (PAs), play a significant role in wine sensory characteristics, specifically bitterness and astringency. Although not consensual, flavan-3-ols and oligomeric PAs are generally considered the primary contributors to wine bitterness. Patatin, a vegetable protein fining agent, has been explored as an alternative to animal and synthetic fining agents for reducing wine bitterness. However, contradictory results exist regarding its effectiveness in removing flavan-3-ols and oligomeric PAs in red wines. In this work, a UPLC-Q-TOF MS/MS method was optimized and validated for accurately measuring flavan-3-ols, as well as dimeric and trimeric PAs, in red wines. The MS/MS analysis of flavan-3-ols, in addition to the typical fragmentation described in the literature, revealed an intense mass fragment resulting from the loss of C3O2 and C3O2 + H2O from the parent ion. It was observed that flavan-3-ols and PAs undergo oxidation during sample preparation, which was reversed by the addition of 5 g/L of ascorbic acid. The method demonstrated good linearity range (2 mg/L to 20 mg/L), detection limit (0.3 mg/L to 0.7 mg/L), quantification limit (0.8 mg/L to 2.2 mg/L), precision (repeatability 2.2% to 7.3%), and accuracy (recovery 98.5% to 100.5%). The application of patatin at different doses (5 g/L to 30 g/L) in two different red wine matrices did not reduce the levels of monomeric, dimeric, and trimeric PAs in red wines. However, similar behaviors were observed for pea protein and gelatin. Therefore, wine fining trials and efficiency measurements of the treatments in each matrix are strongly advised.

Severe Hypertriglyceridemia: A 10-Year Review in a Portuguese Hospital.

INTRODUCTION: Severe hypertriglyceridemia (SHTG) is a rare condition associated with serious complications, such as acute pancreatitis (AP), and the best treatment is still a matter of discussion. The aim of this study is to outline the demographics, management, and outcomes (recurrence and mortality) of complications in patients with SHTG. MATERIAL AND METHODS: A retrospective, observational, and analytical study was carried out by obtaining clinical data from the electronic health records of patients with SHTG admitted to the Internal and Intensive Medicine units from the 1st of January 2009 to the 31st of December 2020 in a university hospital. RESULTS: The cohort included 17 patients. The most common complication was AP (13/17 = 76.5%). Admission to the intensive care unit (ICU) was observed in 84.2%. Among patients with AP, the most commonly administered therapies were insulin (82.4%) and fibrates (76.5%). Plasmapheresis was used in 58.8%, and the criteria for using this technique were mainly based on clinical and laboratory abnormalities. There were no deaths. The readmission rate at 30 days was 36.3%. CONCLUSION: This study shows the morbidity profile associated with SHTG, with a high level of ICU admissions and also a high level of the use of plasmapheresis. In our population, this approach had good results, and this should be highlighted as there are no clear international guidelines for this intervention. Distinguishing between patients with familial chylomicronemia syndrome or with multifactorial chylomicronemia is important as recent specific therapy for lipoprotein lipase (LPL) genetic deficit is available. In the near future, the performance of a genetic study should be considered in patients with SHTG as an attempt to avoid the high recurrence rate of complications of this disease.

Antioxidant and Anti-Inflammatory Activities of Stellera chamaejasme L. Roots and Aerial Parts Extracts.

Natural products, mainly plants, have a crucial role in folk medicine. Particularly, Stellera chamaejasme L. has been traditionally used in Mongolian medicine to treat various diseases, including chronic tracheitis, tuberculosis, and psoriasis. In this study, ethanol (EtOH) and dichloromethane (DCM) extracts of its roots (R) and aerial parts (AP) were evaluated for their antioxidant and anti-inflammatory activities. Thin-layer chromatography demonstrated the presence of flavonoids, namely kaempferol and quercetin-3-O-glucopyranoside, only in the EtOH-AP. Conversely, it showed that kaempferol, quercetin-3-O-glucopyranoside, coumarin, luteolin, rutin, morin, and riboflavin were not present in the other three extracts. The S. chamaejasme extracts exhibited strong antioxidant activity. In addition, the roots extracts presented the highest antioxidant activity against peroxyl radicals, with the EtOH-R being the most potent (IC50 = 0.90 ± 0.07 µg/mL). S. chamaejasme extracts also efficiently inhibited the production of one of the main pro-inflammatory cytokines, interleukin (IL)-6, in a dose-dependent manner by lipopolysaccharide-stimulated macrophages. Particularly, DCM-R was the strongest extract, reducing ≈ 91.5% of the IL-6 production. Since this extract was the most effective, gas chromatography-mass spectrometry (GC-MS) analyses were performed and demonstrated the presence of two fatty acids (palmitic acid and 9-octadecenoic acid), one fatty alcohol (1-hexadecanol), and one triterpenoid (squalene) that can contribute to the observed bioactivity. Herewith, S. chamaejasme extracts, mainly DCM-R, exhibit antioxidant and anti-inflammatory activities that could be applied as new and innovative natural formulations for the treatment of chronic inflammatory diseases.

Supracondylar Apophysis of the Humerus: Rare Cause of High Compression of the Median Nerve / Apófise supracondilar do úmero: Causa rara de compressão alta do nervo mediano

Abstract Supracondylar apophysis (SA) is a bony prominence that originates from the anteromedial aspect of the distal humerus with a lower projection and which, although usually asymptomatic, due to the relationship with adjacent structures can cause symptoms. We describe the case of a 42-year-old woman with pain complaints radiating from her elbow to her hand, with 6 months of evolution. On objective examination, the patient had a sensory deficit in the median nerve territory and decreased grip strength. Radiographs of the distal humerus were performed, in which a bone spike was visible, and magnetic resonance imaging showed thickening of the median nerve epineurium. Electromyography showed severe axonal demyelination of the median nerve proximal to the elbow. A median nerve compression caused by a SA was diagnosed. The patient underwent surgery and, 1 year after the operation, she had a complete clinical recovery. Supracondylar apophysis is a rare, but possible and treatable cause of high median nerve compression.

Resumo A apófise supracondilar (ASC) é uma proeminência óssea que tem origem na face anteromedial do úmero distal com projeção inferior e que, apesar de habitualmente assintomática, pela relação com as estruturas adjacentes pode causar sintomatologia. Descrevemos o caso de uma mulher de 42 anos, com queixas álgicas irradiadas do cotovelo à mão, com 6 meses de evolução. Ao exame objetivo, a paciente apresentava um déficit sensorial no território do nervo mediano e diminuição da força de preensão. Foram realizadas radiografias do úmero distal nas quais era visível uma espícula óssea, e na ressonância magnética era evidente o espessamento do epineuro do nervo mediano. A eletromiografia apresentou uma desmielinização axonal grave do nervo mediano proximal ao cotovelo. Foi diagnosticada uma compressão do nervo mediano por uma ASC. A paciente foi submetida à cirurgia e 1 ano pós-operatório apresentou recuperação clínica total. A ASC é uma causa rara, mas possível e tratável da compressão alta do nervo mediano.

Functional recovery of injured cavernous nerves achieved through endogenous nerve growth factor-containing bioactive fibrous membrane.

Radical prostatectomy is a highly successful treatment for prostate cancer, among the most prevalent manifestations of the illness. Damage of the cavernous nerve (CN) during prostatectomy is the main cause of postoperative erectile dysfunction (ED). In this study, the capability of a personalized bioactive fibrous membrane to regenerate injured CN was investigated. The fibrous membrane bioactivity is conferred by the selectively bound nerve growth factor (NGF) present in the rat urine. In a rat model of bilateral CN crush, the implanted bioactive fibrous membrane induces CN regeneration and restoration of erectile function, showing a significantly increased number of smooth muscle cells and content of endothelial and neuronal nitric oxide synthases (eNOS; nNOS). In addition, the bioactive fibrous membrane promotes nerve regeneration by increasing the number of myelinated axons and nNOS-positive cells, therefore reversing the CN fibrosis found in untreated rats or rats treated with a bare fibrous membrane. Therefore, this personalized regenerative strategy could overcome the recognized drawbacks of currently available treatments for CN injuries. It may constitute an effective treatment for prostate cancer patients suffering from ED after being subject to radical prostatectomy. STATEMENT OF SIGNIFICANCE: The present work introduces a unique strategy to address post-surgical ED resulting from CN injury during pelvic surgery (e.g., radical prostatectomy, radical cystoprostatectomy, abdominoperineal resection). It comprises a bioactive and cell-free fibrous implant, customized to enhance CN recovery. Pre-clinical results in a rat model of bilateral CN crush demonstrated that the bioactive fibrous implant can effectively heal injured CN, and restore penile structure and function. This implant selectively binds NGF from patient fluids (i.e. urine) due to its functionalized surface and high surface area. Moreover, its local implantation reduces adverse side effects. This tailored regenerative approach has the potential to revolutionize the treatment of ED in prostate cancer patients following radical prostatectomy, overcoming current treatment limitations.

Microfluidic Devices: A Tool for Nanoparticle Synthesis and Performance Evaluation.

The use of nanoparticles (NPs) in nanomedicine holds great promise for the treatment of diseases for which conventional therapies present serious limitations. Additionally, NPs can drastically improve early diagnosis and follow-up of many disorders. However, to harness their full capabilities, they must be precisely designed, produced, and tested in relevant models. Microfluidic systems can simulate dynamic fluid flows, gradients, specific microenvironments, and multiorgan complexes, providing an efficient and cost-effective approach for both NPs synthesis and screening. Microfluidic technologies allow for the synthesis of NPs under controlled conditions, enhancing batch-to-batch reproducibility. Moreover, due to the versatility of microfluidic devices, it is possible to generate and customize endless platforms for rapid and efficient in vitro and in vivo screening of NPs' performance. Indeed, microfluidic devices show great potential as advanced systems for small organism manipulation and immobilization. In this review, first we summarize the major microfluidic platforms that allow for controlled NPs synthesis. Next, we will discuss the most innovative microfluidic platforms that enable mimicking in vitro environments as well as give insights into organism-on-a-chip and their promising application for NPs screening. We conclude this review with a critical assessment of the current challenges and possible future directions of microfluidic systems in NPs synthesis and screening to impact the field of nanomedicine.

Primary Splenic Diffuse Large B-Cell Lymphoma: A Case Report.

Primary splenic lymphoma (PSL) is a rare disease and an improbable cause of splenomegaly or splenic nodules. On the contrary, splenic secondary involvement as part of an advanced lymphoproliferative disorder is more common. The authors present the case of a 49-year-old woman with a primary splenic diffuse large B-cell lymphoma (PS-DLBCL), in which the absence of other organs' involvement determined an ultrasound-guided biopsy of the spleen to achieve a definitive diagnosis. With this case the authors intend to emphasise the extensive differential diagnosis of splenomegaly, splenic nodules or infiltrates, the usefulness of splenic biopsy in establishing the diagnosis and recall a rare disease, with non-specific presenting symptoms, in which the diagnostic workup is challenging. LEARNING POINTS: The differential diagnosis of splenic nodules or infiltrates is vast and challenging, and it includes haematological diseases, systemic infectious diseases but also non-malignant infiltrative diseases.Although some lymphomas frequently present with splenomegaly, this is not the case of DLBCL, with the exception of PS-DLBCL.PS-DLBCL is a very rare pathology, accounting for 1% of all DLBCL and less than 1% of all NHL.

Combination Therapy With CD147-Targeted Nanoparticles Carrying Phenformin Decreases Lung Cancer Growth.

Lung cancer is one of the most fatal cancers worldwide. Resistance to conventional therapies remains a hindrance to patient treatment. Therefore, the development of more effective anti-cancer therapeutic strategies is imperative. Solid tumors exhibit a hyperglycolytic phenotype, leading to enhanced lactate production; and, consequently, its extrusion to the tumor microenvironment. Previous data reveals that inhibition of CD147, the chaperone of lactate transporters (MCTs), decreases lactate export in lung cancer cells and sensitizes them to phenformin, leading to a drastic decrease in cell growth. In this study, the development of anti-CD147 targeted liposomes (LUVs) carrying phenformin is envisioned, and their efficacy is evaluated to eliminate lung cancer cells. Herein, the therapeutic effect of free phenformin and anti-CD147 antibody, as well as the efficacy of anti-CD147 LUVs carrying phenformin on A549, H292, and PC-9 cell growth, metabolism, and invasion, are evaluated. Data reveals that phenformin decreases 2D and 3D-cancer cell growth and that the anti-CD147 antibody reduces cell invasion. Importantly, anti-CD147 LUVs carrying phenformin are internalized by cancer cells and impaired lung cancer cell growth in vitro and in vivo. Overall, these results provide evidence for the effectiveness of anti-CD147 LUVs carrying phenformin in compromising lung cancer cell aggressiveness.

Synthesis and Anti-Inflammatory Evaluation of a Library of Chiral Derivatives of Xanthones Conjugated with Proteinogenic Amino Acids.

In recent decades, the relationship between drug chirality and biological activity has been assuming enormous importance in medicinal chemistry. Particularly, chiral derivatives of xanthones (CDXs) have interesting biological activities, including enantioselective anti-inflammatory activity. Herein, the synthesis of a library of CDXs is described, by coupling a carboxyxanthone (1) with both enantiomers of proteinogenic amino esters as chiral building blocks (2-31), following the chiral pool strategy. The coupling reactions were performed at room temperature with good yields (from 44 to 99.9%) and very high enantiomeric purity, with most of them presenting an enantiomeric ratio close to 100%. To afford the respective amino acid derivatives (32-61), the ester group of the CDXs was hydrolyzed in mild alkaline conditions. Consequently, in this work, sixty new derivatives of CDXs were synthetized. The cytocompatibility and anti-inflammatory activity in the presence of M1 macrophages were studied for forty-four of the new synthesized CDXs. A significant decrease in the levels of a proinflammatory cytokine targeted in the treatment of several inflammatory diseases, namely interleukin 6 (IL-6), was achieved in the presence of many CDXs. The amino ester of L-tyrosine (X1AELT) was the most effective in reducing IL-6 production (52.2 ± 13.2%) by LPS-stimulated macrophages. Moreover, it was ≈1.2 times better than the D-enantiomer. Indeed, enantioselectivity was observed for the majority of the tested compounds. Thus, their evaluation as promising anti-inflammatory drugs should be considered.

Moyamoya Disease and Syndrome in Caucasian Patients.

Moyamoya disease is a unique cerebrovascular disease characterized by narrowing of the terminal portion of internal carotid arteries and circle of Willis, with consequent development of a network of collateral vessels in response to brain ischemia. Moyamoya vascular pattern can be idiopathic (Moyamoya disease), is more likely to occur in individuals of Asian ascent and in the pediatric age, or is associated with other diseases (Moyamoya syndrome). We present two cases of stroke in young adults, where workup revealed Moyamoya-type vascular changes. The first case report is of a 42-year-old woman presenting with hemorrhagic stroke, with classic angiographic findings of Moyamoya disease, otherwise asymptomatic. The second case concerns a 36-year-old woman admitted with ischemic stroke; besides the typical angiographic pattern of Moyamoya, the patient was diagnosed with antiphospholipid antibody syndrome and Graves' disease, two conditions known to be associated with this vasculopathy. These case reports illustrate the need to consider this entity in the etiological evaluation of ischemic and hemorrhagic cerebrovascular events, even in Western countries, since management and secondary prevention require specific approaches.

Size-Dependent Polymeric Nanoparticle Distribution in a Static versus Dynamic Microfluidic Blood Vessel Model: Implications for Nanoparticle-Based Drug Delivery.

Nanoparticles (NPs) have been widely investigated in the nanomedicine field. One of the main challenges is to accurately predict the NP distribution and fate after administration. Microfluidic platforms acquired huge importance as tools to model the in vivo environment. In this study, we leveraged a microfluidic platform to produce FITC-labeled poly(lactide-co-glycolide)-block-poly(ethylene glycol) (PLGA-PEG) NPs with defined sizes of 30, 50, and 70 nm. The study aimed to compare the ability of NPs with differences of 20 nm in size to cross an endothelial barrier using static (Transwell inserts) and dynamic (microfluidic perfusion device) in vitro models. Our results evidence a size-dependent NP crossing in both models (30 > 50 > 70 nm) and highlight the bias deriving from the static model, which does not involve shear stresses. The permeation of each NP size was significantly higher in the static system than in the dynamic model at the earliest stages. However, it gradually decreased to levels comparable with those of the dynamic model. Overall, this work highlights clear differences in NP distribution over time in static versus dynamic conditions and distinct size-dependent patterns. These findings reinforce the need for accurate in vitro screening models that allow for more accurate predictions of in vivo performance.

The Influence of Feeder Cell-Derived Extracellular Matrix Density on Thymic Epithelial Cell Culture.

The thymus is responsible for the selection and development of T cells, having an essential role in the establishment of adaptive immunity. Thymic epithelial cells (TECs) are key players in T cell development interacting with thymocytes in the thymic 3D environment. Feeder-layer cells have been frequently used as platforms for the successful establishment of TEC cultures. Nevertheless, the role of the feeder cell-derived extracellular matrix (ECM) on TEC cultures was not previously reported. Therefore, this work aimed at assessing the effect of the ECM produced by feeder cells cultured at two different densities on the establishment of TEC culture. Due to the high surface area and porosity, electrospun fibrous meshes were used to support ECM deposition. The feeder cell-derived ECM was efficiently recovered after decellularization, maintaining the composition of major proteins. All the decellularized matrices were permeable and showed an increase in surface mechanical properties after decellularization. TEC cultures confirmed that the ECM density impacts cellular performance, with higher densities showing a decreased cellular activity. Our findings provide evidence that feeder cell-derived ECM is a suitable substrate for TEC culture and can potentially be applied in thymus bioengineering.

Long non-coding RNA H19 regulates matrisome signature and impacts cell behavior on MSC-engineered extracellular matrices.

BACKGROUND: The vast and promising class of long non-coding RNAs (lncRNAs) has been under investigation for distinct therapeutic applications. Nevertheless, their role as molecular drivers of bone regeneration remains poorly studied. The lncRNA H19 mediates osteogenic differentiation of Mesenchymal Stem/Stromal Cells (MSCs) through the control of intracellular pathways. However, the effect of H19 on the extracellular matrix (ECM) components is still largely unknown. This research study was designed to decode the H19-mediated ECM regulatory network, and to reveal how the decellularized siH19-engineered matrices influence MSC proliferation and fate. This is particularly relevant for diseases in which the ECM regulation and remodeling processes are disrupted, such as osteoporosis. METHODS: Mass spectrometry-based quantitative proteomics analysis was used to identify ECM components, after oligonucleotides delivery to osteoporosis-derived hMSCs. Moreover, qRT-PCR, immunofluorescence and proliferation, differentiation and apoptosis assays were performed. Engineered matrices were decellularized, characterized by atomic force microscopy and repopulated with hMSC and pre-adipocytes. Clinical bone samples were characterized by histomorphometry analysis. RESULTS: Our study provides an in-depth proteome-wide and matrisome-specific analysis of the ECM proteins controlled by the lncRNA H19. Using bone marrow-isolated MSC from patients with osteoporosis, we identified fibrillin-1 (FBN1), vitronectin (VTN) and collagen triple helix repeat containing 1 (CTHRC1), among others, as having different pattern levels following H19 silencing. Decellularized siH19-engineered matrices are less dense and have a decreased collagen content compared with control matrices. Repopulation with naïve MSCs promotes a shift towards the adipogenic lineage in detriment of the osteogenic lineage and inhibits proliferation. In pre-adipocytes, these siH19-matrices enhance lipid droplets formation. Mechanistically, H19 is targeted by miR-29c, whose expression is decreased in osteoporotic bone clinical samples. Accordingly, miR-29c impacts MSC proliferation and collagen production, but does not influence ALP staining or mineralization, revealing that H19 silencing and miR-29c mimics have complementary but not overlapping functions. CONCLUSION: Our data suggest H19 as a therapeutic target to engineer the bone ECM and to control cell behavior.