Staff member

De Chiara, Francesco

JTD

Fibroadipogenic precursor cells (FAPs) are key contributors to the fibrotic and adipogenic remodeling observed in Duchenne muscular dystrophy (DMD), yet their precise role in muscle degeneration remains unclear. In this study, we investigated how FAPs from DMD muscle influence myogenesis by conducting co-culture experiments using healthy myoblasts with FAPs derived from either healthy or DMD biopsies, in both direct and indirect contact conditions. We observed that healthy FAPs enhance myogenic differentiation, increasing myotube area, while DMD FAPs significantly inhibit it. To identify underlying molecular mechanisms, we performed mass spectrometry of the FAP secretome and found that 368 of 760 detected proteins were upregulated in DMD FAPs, including C4b-binding protein alpha chain (C4BPA), which showed a notable increase. In vitro treatment of myoblasts with recombinant C4BPA led to severe impairment of myotube formation, evidenced by reduced myotube area, fewer nuclei per myotube, and smaller myotube size. C4BPA exposure also downregulated myogenic markers and upregulated genes associated with muscle atrophy. These findings were further validated in a 3D engineered muscle model, where C4BPA significantly reduced contractile function. Importantly, silencing C4BPA in DMD-derived cultures partially restored myogenic capacity, improving both the differentiation index and nuclear content per myotube. Together, our data demonstrate that DMD FAPs exert anti-myogenic effects, at least in part, through elevated secretion of C4BPA, which interferes with muscle differentiation and function. This highlights C4BPA as a novel effector of muscle degeneration and a promising therapeutic target for modulating the fibrotic environment in DMD. Targeting specific secreted factors from pathological FAPs may help preserve muscle regeneration and mitigate disease progression in dystrophic muscles.

JTD

Organ-on-chip (OoC) systems are advancing rapidly as physiologically relevant in vitro models. However, real-time, noninvasive metabolic monitoring tools remain lacking. While NMR offers a powerful solution, current setups are not compatible with the planar format of microfluidic chips. Here we introduce the first benchtop NMR spectrometer for real-time metabolic monitoring of cell cultures on microfluidic platforms, utilizing hyperpolarization via dissolution dynamic nuclear polarization. This work details modifications made to a commercial benchtop NMR spectrometer, including the design and fabrication of a microfluidic platform that enables precise injection of hyperpolarized substrates and continuous renewal of cell culture media. The platform integrates a radiofrequency coil for signal transmission and reception and incorporates a custom-built sample carrier. Preliminary NMR data acquired with this system demonstrate its feasibility for dynamic metabolic studies.

JTD Keywords: Cancer, Pyruvate

Electrical pulse stimulation (EPS) is used to replicate motor neuron activation in muscle tissues, enabling in vitro studies of muscle contraction. However, both custom-built and commercial existing EPS systems often suffer from significant limitations, including limited scalability, high cost, and lack of flexibility for experimental adaptation. This work presents the Myo-MOVES platform, a practical solution for stimulating 3D skeletal muscle tissues. The device has been designed as an intuitive EPS system consisting of two main components: a selector and a stimulator that adapts to commercial 24-well culture plates. The Myo-MOVES selector enables targeted stimulation of single or multiple wells, while the stimulator delivers electrical signals via graphite electrodes to the plate containing 3D skeletal muscle samples. The Myo-MOVES platform was technically validated and employed as a proof of concept to investigate sarcolemmal damage induced by muscle contraction in Duchenne muscular dystrophy (DMD) 3D skeletal muscle tissues. Taking advantage of the versatility of the device, we validated Myo-MOVES through the assessment of force generation in DMD engineered muscle tissues and the detection of contraction-induced sarcolemmal damage via Evans blue dye uptake and the release of creatine kinase (CK), the gold standard marker of muscle damage. These findings demonstrate the feasibility of using Myo-MOVES to induce and study functionally relevant disease phenotypes in DMD 3D skeletal muscle tissues. These results highlight the system's potential as a valuable tool for future applications in the field of 3D skeletal muscle tissue engineering, including drug screening and the study of DMD therapies and other muscular diseases.

JTD

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and Long Covid-19 (LC-19) are complex conditions with no diagnostic markers or consensus on disease progression. Despite extensive research, no in vitro model exists to study skeletal muscle wasting, peripheral weakness, or potential therapies. We developed 3D in vitro skeletal muscle tissues to map muscle adaptations to patient sera over time. Short exposures (48 H) to patient sera led to a significant reduction in muscle contractile strength. Transcriptomic analysis revealed the upregulation of protein translation, glycolytic enzymes, disturbances in calcium homeostasis, hypertrophy, and mitochondrial hyperfusion. Structural analyses confirmed myotube hypertrophy and elevated mitochondrial oxygen consumption In ME/CFS. While muscles initially adapted by increasing glycolysis, prolonged exposure (96-144 H) caused muscle fragility and weakness, with mitochondria fragmenting into a toroidal conformation. We propose that skeletal muscle tissue in ME/CFS and LC-19 progresses through a hypermetabolic state, leading to severe muscular and mitochondrial deterioration. This is the first study to suggest such transient metabolic adaptation.

JTD Keywords: Chronic fatigue syndrome, Long covid-19, Metabolic adaptatio, Skeletal muscle

Microscopy is a fundamental tool in biological research. However, conventional microscopes require manual operation and depend on user and equipment availability, limiting their suitability for continuous observation. Moreover, their size and complexity make them impractical for in situ experimentation. In this work, we present a novel, compact, affordable, and portable microscope that enables continuous in situ monitoring by being placed directly on biological samples. This chip-sized lensless holographic microscope (CLHM) is specifically designed to overcome the limitations of traditional microscopy. The device consists solely of an ultra-compact, state-of-the-art micro-LED display and a CMOS sensor, all enclosed within a 3D-printed housing. This unique light source enables a size that is markedly smaller than any comparable technology, allowing a resolution of 2.19 mu m within a 7 mm distance between the light source and the camera. This paper demonstrates the CLHM's versatility by monitoring in vitro models and performing whole-organism morphological analyses of small specimens. These experiments underscore its potential as an on-platform sensing device for continuous, in situ biological monitoring across diverse models.

JTD Keywords: Angiogenesis, Compact microscope, Fermentation, Holography, Lab-on-a-chip, Lensless, Real-time monitoring, Zebrafish

Ramón-Azcón, J.

JTD

JTD Keywords: Industry, innovation and infrastructure

Laminopathies are causally associated with mutations on the Lamin A/C gene (LMNA). To date, more than 400 mutations in LMNA have been reported in patients. These mutations are widely distributed throughout the entire gene and are associated with a wide range of phenotypes. Unfortunately, little is known about the mechanisms underlying the effect of the majority of these mutations. This is the case of more than 40 mutations that are located at exon 4. Using CRISPR/Cas9 technology, we generated a collection of Lmna exon 4 mutants in mouse C2C12 myoblasts. These cell models included different types of exon 4 deletions and the presence of R249W mutation, one of the human variants associated with a severe type of laminopathy, LMNA-associated congenital muscular dystrophy (L-CMD). We characterized these clones by measuring their nuclear circularity, myogenic differentiation capacity in 2D and 3D conditions, DNA damage, and levels of p-ERK and p-AKT (phosphorylated Mitogen-Activated Protein Kinase 1/3 and AKT serine/threonine kinase 1). Our results indicated that Lmna exon 4 mutants showed abnormal nuclear morphology. In addition, levels and/or subcellular localization of different members of the lamin and LINC (LInker of Nucleoskeleton and Cytoskeleton) complex were altered in all these mutants. Whereas no significant differences were observed for ERK and AKT activities, the accumulation of DNA damage was associated to the Lmna p.R249W mutant myoblasts. Finally, significant myogenic differentiation defects were detected in the Lmna exon 4 mutants. These results have key implications in the development of future therapeutic strategies for the treatment of laminopathies.

JTD Keywords: CRISPR, Laminopathy, LMNA, Nuclear envelope

Non-alcoholic fatty liver (NAFLD) is a metabolic disorder related to a chronic lipid accumulation within the hepatocytes. This disease is the most common liver disorder worldwide, and it is estimated that it is present in up to 25% of the world’s population. However, the real prevalence of this disease and the associated disorders is unknown mainly because reliable and applicable diagnostic tools are lacking. It is known that the level of albumin, a pleiotropic protein synthesized by hepatocytes, is correlated with the correct function of the liver. The development of a complementary tool that allows direct, sensitive, and label-free monitoring of albumin secretion in hepatocyte cell culture can provide insight into NAFLD’s mechanism and drug action. With this aim, we have developed a simple integrated plasmonic biosensor based on gold nanogratings from periodic nanostructures present in commercial Blu-ray optical discs. This sensor allows the direct and label-free monitoring of albumin in a 2D fatty liver disease model under flow conditions using a highly-specific polyclonal antibody. This technology avoids both the amplification and blocking steps showing a limit of detection within pM range (≈0.26 ng/mL). Thanks to this technology, we identified the optimal fetal bovine serum (FBS) concentration to maximize the cells’ lipid accumulation. Moreover, we discovered that the hepatocytes increased the amount of albumin secreted on the third day from the lipids challenge. These data demonstrate the ability of hepatocytes to respond to the lipid stimulation releasing more albumin. Further investigation is needed to unveil the biological significance of that cell behavior.

JTD Keywords: 2D fatty liver in vitro model, Blu-Ray disc, Plasmonic nanomaterials, Label-Free Biosensing

Currently, the fabrication of scaffolds for engineered skeletal muscle tissue is unable to reach the millimeter size. The main drawbacks are the poor nutrients diffusion, lack of internal structure to align precursor cells as well as poor mechanical and electric properties. Herein, we present a combination of gelatin-carboxymethyl cellulose materials polymerised by a cryogelation process that allowed us to reach scaffold fabrication up to millimeters size and solve the main problems related with large size muscle tissue constructs. 1) By incorporating carbon nanotubes (CNT) we can improve the electrical properties of the scaffold, thereby enhancing tissue maturation when applying an electric pulse stimulus (EPS). 2) We have fabricated an anisotropic internal three-dimensional microarchitecture pore distribution with high aligned morphology to enhance cells alignment, cell fusion and myotubes formation. With this set up, we were able to generate a fully functional skeletal muscle tissue using a combination of EPS and our doped-biocomposite scaffold and obtain a mature tissue in a millimeter scale. We also characterized pore distribution, swelling, stiffness and conductivity of the scaffold. Moreover, we proved that the cells are viable and able to fuse in a three-dimensional (3D) functional myotubes throughout the scaffold. In conclusion, we fabricate a biocompatible and customizable scaffold for 3D cell culture suitable for a wide range of application such as organ-on-a-chip, drug screening, transplantation and disease modelling.

JTD

Understanding the protein-secretion dynamics from single, specific tissues is critical toward the advancement of disease detection and treatments. However, such secretion dynamics remain difficult to measure in vivo due to the uncontrolled contributions from other tissue populations. Here, we describe an integrated platform designed for the reliable, near real-time measurements of cytokines secreted from an in vitro single-tissue model. In our setup, we grow 3D biomimetic tissues to discretize cytokine source, and we separate them from a magnetic microbead-based biosensing system using a Transwell insert. This design integrates physiochemically controlled biological activity, high-sensitivity protein detection (LOD < 20 pg mL−1), and rapid protein diffusion to enable non-invasive, near real-time measurements. To showcase the specificity and sensitivity of the system, we use our setup to probe the inflammatory process related to the protein Interleukine 6 (IL-6) and to the Tumor Necrosis Factor (TNF-α). We show that our setup can monitor the time-dependence profile of IL-6 and TNF-α secretion that results from the electrical and chemical stimulation of 3D skeletal muscle tissues. We demonstrate a novel and affordable methodology for discretizing the secretion kinetics of specific tissues for advancing metabolic-disorder studies and drug-screening applications.

JTD Keywords: Microphysiological tissues, Tissue engineering, Electrochemical, biosensors, Magnetic particles, Skeletal muscle, Electric stimulation

Despite the increasing number of organs-on-a-chip that have been developed in the past decade, limited efforts have been made to integrate a sensing system for in situ continual measurements of biomarkers from three-dimensional (3D) tissues. Here, we present a custom-made integrated platform for muscle cell stimulation under fluidic conditions connected with a multiplexed high-sensitivity electrochemical sensing system for in situ monitoring. To demonstrate this, we use our system to measure the release levels and release time of interleukin 6 and tumor necrosis factor alpha in vitro by 3D muscle microtissue under electrical and biological stimulations. Our experimental design has enabled us to perform multiple time point measurements using functionalized screen-printed gold electrodes with sensitivity in the ng mL−1 range. This affordable setup is uniquely suited for monitoring factors released by 3D single cell types upon external stimulation for metabolic studies.

JTD

Non-alcoholic fatty liver disease (NAFLD) is characterized by lipid accumulation within the liver affecting 1 in 4 people worldwide. As the new silent killer of the twenty-first century, NAFLD impacts on both the request and the availability of new liver donors. The liver is the first line of defense against endogenous and exogenous metabolites and toxins. It also retains the ability to switch between different metabolic pathways according to food type and availability. This ability becomes a disadvantage in obesogenic societies where most people choose a diet based on fats and carbohydrates while ignoring vitamins and fiber. The chronic exposure to fats and carbohydrates induces dramatic changes in the liver zonation and triggers the development of insulin resistance. Common believes on NAFLD and different diets are based either on epidemiological studies, or meta-analysis, which are not controlled evidences; in most of the cases, they are biased on test-subject type and their lifestyles. The highest success in reverting NAFLD can be attributed to diets based on high protein instead of carbohydrates. In this review, we discuss the impact of NAFLD on body metabolic plasticity. We also present a detailed analysis of the most recent studies that evaluate high-protein diets in NAFLD with a special focus on the liver and the skeletal muscle protein metabolisms.

JTD Keywords: High protein diet, Low carbohydrates, NAFLD, NASH, Physical activity

Concentrations down to 3 nM of the rhS100A4 protein, associated with human tumor development, have been detected in undiluted urine using an integrated sensor based on microring resonators in the emerging Al2O3 photonic platform. The fabricated microrings were designed for operation in the C-band (λ = 1565 nm) and exhibited a high-quality factor in air of 3.2 × 105. The bulk refractive index sensitivity of the devices was ~100 nm/RIU (for TM polarization) with a limit of detection of ~10−6 RIU. A surface functionalization protocol was developed to allow for the selective binding of the monoclonal antibodies designed to capture the target biomarker to the surface of the Al2O3 microrings. The detection of rhS100A4 proteins at clinically relevant concentrations in urine is a big milestone towards the use of biosensors for the screening and early diagnosis of different cancers. Biosensors based on this microring technology can lead to portable, multiplexed and easy-to-use point of care devices.

JTD Keywords: Distributed feedback lasers, Effective refractive index, Laser coupling, Polarization maintaining fibers, Refractive index, Scanning electron microscopy

Abstract New biocompatible materials have enabled the direct 3D printing of complex functional living tissues, such as skeletal and cardiac muscle. Gelatinmethacryloyl (GelMA) is a photopolymerizable hydrogel composed of natural gelatin functionalized with methacrylic anhydride. However, it is difficult to obtain a single hydrogel that meets all the desirable properties for tissue engineering. In particular, GelMA hydrogels lack versatility in their mechanical properties and lasting 3D structures. In this work, a library of composite biomaterials to obtain versatile, lasting, and mechanically tunable scaffolds are presented. Two polysaccharides, alginate and carboxymethyl cellulose chemically functionalized with methacrylic anhydride, and a synthetic material, such as poly(ethylene glycol) diacrylate are combined with GelMA to obtain photopolymerizable hydrogel blends. Physical properties of the obtained composite hydrogels are screened and optimized for the growth and development of skeletal muscle fibers from C2C12 murine cells, and compared with pristine GelMA. All these composites show high resistance to degradation maintaining the 3D structure with high fidelity over several weeks. Altogether, in this study a library of biocompatible novel and totally versatile composite biomaterials are developed and characterized, with tunable mechanical properties that give structure and support myotube formation and alignment.

JTD

Animal models have been the main resources for drug discovery and prediction of drugs’ pharmacokinetic responses in the body. However, noticeable drawbacks associated with animal models include high cost, low reproducibility, low physiological similarity to humans, and ethical problems. Engineered tissue models have recently emerged as an alternative or substitute for animal models in drug discovery and testing and disease modeling. In this review, we focus on skeletal muscle and cardiac muscle tissues by first describing their characterization and physiology. Major fabrication technologies (i.e., electrospinning, bioprinting, dielectrophoresis, textile technology, and microfluidics) to make functional muscle tissues are then described. Finally, currently used muscle tissue models in drug screening are reviewed and discussed.

JTD Keywords: Cardiac muscle, Drug screening, Engineering muscle, Human pharmacological response, Physiological similarity, Skeletal muscle

Tissue engineering (TE) aims to restore function or replace damaged tissue through biological principles and engineering. Nanofibers are attractive substrates for tissue regeneration applications because they structurally mimic the native extracellular matrix. Composite nanofibers, which are hybrid nanofibers blended from natural and synthetic polymers, represent a major advancement in TE and regenerative medicine, since they take advantage of the physical properties of the synthetic polymer and the bioactivity of the natural polymer while minimizing the disadvantages of both. Although various nanofibrous matrices have been applied to almost all the areas of TE, in this chapter we will focus on nanofiber composites scaffolds for vascular TE.

JTD Keywords: Blood vessels, Nanofiber composite, Tissue engineering, Vascularized tissue