Biology subjects

Moroni, L.

Publications and source records attributed to Moroni, L..

At least 19 recordsLinked to original sources

bioRxiv2026

Marrow-mimicry MEW scaffolds recapitulate erythroblastic islands for high-density cRBC biomanufacturing

Biomanufactured cell therapies are delivering a step-change in modern medicine, but their high costs have prevented widespread adoption. Cultured red blood cells (cRBCs) are one type of cell therapy in clinical trials for off-the-shelf blood transfusion but remain limited by the exorbitant costs of manufacturing trillions of RBCs required for a blood transfusion. These costs could be mitigated if culture platforms could grow cells at higher density, like those found in the bone marrow, requiring less media and lower concentrations of expensive growth factors per cell. We engineered melt electrowritten (MEW) polycaprolactone (PCL) scaffolds in geometries which imitate marrow trabeculae structures and were then plasma-treated to enable peripheral blood mononuclear cells (PB MNC) attachment. PB MNC monocytes first adhered to the scaffold and rapidly differentiated into CD68+ macrophage-like cells. These macrophage-loaded scaffolds adhered CD71+ erythroblasts, which then grew from 10 to 25% between days 4 and 8 and produced CD235a+ erythroid cells by day 20, under erythrogenic culture conditions. MEW PCL scaffolds in trabecular-like geometries had greater surface area, adhered more macrophages, and generated more erythroblasts than traditional MEW crosshatch geometries. The MEW scaffolds continuously released less-adherent cells to the supernatant, providing a 74% increase in supernatant cell numbers versus liquid suspension controls with identical cell seeding numbers and media volumes. High-cell-density scaffolds exhibited increased metabolism and endogenous production of self-supportive cytokines, which may offer cost-saving opportunities through future media optimisation. This study reports marrow trabeculae-mimetic MEW PCL designs capable of selectively adhering PB MN cells to develop macrophage-coated scaffolds, which provided physical and paracrine support for cRBC production. This high-cell-density culture platform may be useful for future cell therapy biomanufacturing protocols.

bioengineering↗

bioRxiv2026

The hydrocup: a hollow electrospun scaffold for in vivo hydrogel-laden cell delivery

For regenerative medicine applications, transplanted cells are difficult to retain in a specific place to maximize the localized release of cytokines, which can positively modulate immune responses, angiogenesis and tissue regeneration. Here, we developed a hollow electrospun (ESP) scaffold with one open and one closed end to enclose a hydrogel laden with human mesenchymal stem cells (hMSCs); we termed this combination the hydrocup. hMSCs remained viable for 28 days in vitro and released functional cytokines similar to hMSCs in transplanted but non-enclosed hydrogels. The hydrocups remained intact and fixed in place for 6 weeks after subcutaneous implantation in rats, demonstrating in vivo compatibility. These results demonstrate that the hydrocup is the first, to our knowledge, cell-laden hydrogel delivery scaffold. This innovation could be developed for a wide variety of cell-based cytokine secretion or drug-laden hydrogel applications.

bioengineering↗

bioRxiv2026

ReCardioids: a robust human heart organoid platform to model doxorubicin- and radiotherapy-induced cardiotoxicity

As global cancer incidence rises, so does the population of cancer survivors, making long-term and post-treatment quality of life a central clinical concern. Despite modern improvements in treatment, cardiotoxic side effects caused by anthracycline agents and thoracic radiotherapy remain a major clinical challenge. Human iPSC-derived heart organoids offer a promising alternative to animal and 2D in vitro models, but their utility is limited by inter-organoid variability. To address this, we developed reCardioids, a robust heart organoid model generated by dissociation and reaggregation of self-assembling cardioids. Through single-cell transcriptomics, we demonstrated that reCardiods maintain cellular diversity while also exhibiting a more mature cardiomyocyte phenotype compared to non-dissociated cardioids. To validate their utility as a preclinical in vitro model, we evaluated their response after exposure to doxorubicin and clinically relevant doses of {gamma}-radiation. reCardioids successfully modeled doxorubicin-induced cytotoxicity, metabolic decline and altered contractile dynamics. Bulk RNA sequencing following radiation exposure revealed a temporal trajectory of injury, progressing from acute DNA damage through vascular stunting, metabolic dysfunction and eventually compensatory pathological hypertrophic remodeling.

bioengineering↗

bioRxiv2026

Microphysiological Flow Batteries For Dynamic EDC Screening Of mESC-derived Thyroid Organoids

Endocrine disrupting chemicals (EDCs) are ubiquitous environmental contaminants capable of dysregulating the production of thyroid hormones. Traditional thyroid toxicological assays rely on 2D cell cultures and animal models, both of which fail to accurately recapitulate human thyroid physiology and provide limited mechanistic insight into EDC toxicity. To overcome these limitations, we report a novel thyroid-on-chip platform integrating mouse embryonic stem cell-derived thyroid organoids with advanced organ-on-chip (OoC) technology and downstream multi-omics analysis. The platform leverages a reversibly-sealed microphysiological flow battery (MFB) to allow scale up of dynamic organoid culture and controlled chemical exposure while reducing operational complexity. Upon EDC exposure, transcriptomic and proteomic analysis revealed new molecular signatures of thyroid disruption across four different EDC classes, even at very low EDC concentrations (1nM), validating the capacity of this system to mechanistically dissect EDC-induced responses. This represents an integrated platform consists of an advanced physiologically relevant assay framework for next-generation endocrine toxicity testing, bridging the gap between in vitro screening and in vivo thyroid physiology.

bioengineering↗

bioRxiv2026

Engineering a hybrid 3D construct for bone regeneration to promote simultaneous pre-vascularization and osteogenic differentiation in vitro

Critical-sized bone defects represent a challenge in bone tissue engineering, due to insufficient vascularization that results in implant failure. Scaffold pre-vascularization is a promising strategy to create a functional microvascular network that integrates with host vasculature. In this study, we present a hybrid 3D construct comprising a hyaluronic acid-based hydrogel and a 3D printed polycaprolactone/{beta}-tricalcium phosphate scaffold, to support vascular network formation and osteogenic differentiation. Peptide-functionalized (i.e. RGD, YIGSR, IKVAV, QK) hydrogels were obtained via thiol-ene chemistry, using two crosslinkers (PEG-diSH or MMP-diSH). Preliminary biological experiments assessed human mesenchymal stromal cells (hMSCs), endothelial cells (hUVECs), and their co-culture, on different gel formulations. All cell conditions displayed enhanced spreading and metabolic activity on gel formulations comprising RGD; thus these (i.e. RGD only and a combination of RGD/YIGSR) were selected for further studies. Cells were then mixed with the hydrogel precursor solutions, which were injected to embed the scaffolds and crosslinked using a UV lamp. After 7 days, tubule formation was observed only in co-culture conditions, highlighting the importance of cellular crosstalk for the formation of a vascular network. Significant differences were found across the tested formulations. In the RGD-PEG constructs, hUVECs formed tubule-like structures, surrounded by hMSCs, exhibiting pericyte-like behavior, supported by the upregulation of SMA gene. Conversely, in the RGD/YIGSR-MMP conditions, hMSCs were mostly located on the scaffold fibers, and showed the highest expression of early osteogenic markers (RUNX2 and ALP). Overall, we demonstrated that the hybrid system with tailored hydrogel chemistry can support simultaneous microvascular organization and osteogenic commitment, offering a promising platform for bone tissue engineering applications. However, further studies involving longer culture periods will aim at clarifying the complex interplay between material composition, cell crosstalk and spatial organization and their influence on the maturation and stability of the vascular network.

bioengineering↗

bioRxiv2026

Microfluidic bioprinting of a physiologically relevant thyroid three-dimensional in vitro model

Endocrine disruptors (EDs) are an exogenous group of compounds associated with thyroid malfunctioning in the human body. Nonetheless, there are currently no adequate in vivo or in vitro models for the preclinical testing of these compounds since both animal and two-dimensional (2D) cell-based models are not able to mimic thyroid physiological conditions from both functional and three-dimensional (3D) organization perspective. Recently, bioprinting technologies emerged as an innovative tool in the field of regenerative medicine and advanced 3D in vitro models that allow the creation of 3D well-organized structures able to mirror physiologically relevant tissue and organ architectures. In this study, we evaluated microfluidic bioprinting as a biofabrication technology to develop a 3D in vitro model of the thyroid gland. We studied the fundamental parameters to obtain a fine control over the bioprinted fibres for different biomaterials. Then, we assessed the possibility to bioprint single thyroid cells, thyroid spheroids and finally mouse embryonic stem cell-derived thyroid follicles. The different cell types maintained high viability and metabolic activity. The bioprinted thyroid model showed high expression of different early and late functional markers and to be responsive to ED exposure. These bioprinted thyroid constructs could provide a new set of advanced 3D in vitro models to test potential EDs and possible adverse outcomes that may be associated with their administration or exposure.

bioengineering↗

bioRxiv2026

Decellularized Small Intestine for Full Thickness Burn Wound Treatment

Treating extensive full-thickness burn wounds remains difficult in clinical practice because available donor skin is often limited, the risk of infection is high, and many standard dressings do not perform well when defects are large or structurally complex. These limitations have shifted attention to decellularized extracellular matrix (dECM) scaffolds, which can provide physical coverage while preserving biochemical cues that may support tissue repair. Based on this rationale, we designed a decellularization method that improves reagent penetration to produce a full-thickness porcine decellularized small intestine (dSI) scaffold for use in burn wound coverage. The protocol removed most cellular material while leaving low levels of detergent residue, and it maintained the native three-layer structure of the intestinal wall. Most key ECM components, such as collagen and glycosaminoglycans, were also retained. In this study, the dSI showed several properties relevant to burn care, capacity to absorb large amounts of fluid, water vapor transmission rates similar to those reported for skin, and resisted microbial penetration in vitro. From a mechanical standpoint, the scaffold retained anisotropic behaviour and remained stable under cyclic loading. This pattern indicates that it could withstand repeated deformation instead of acting like a fragile membrane. Degradation tests under enzymatic and oxidative conditions indicate that the material breaks down in a controlled way over a period that appears consistent with typical wound-healing timelines. In vitro assays indicated that the scaffold was cytocompatible, as human dermal fibroblasts and keratinocytes both attached to its surface and continued to proliferate. Cell responses differed depending on surface orientation, suggesting that preserved intestinal layers may shape cell behaviour in ways that are often missing in thinner or more uniform matrices. Overall, full-thickness dSI appears to act as a biologically active scaffold and shows mechanical properties that exceed those of many currently used burn dressings.

bioengineering↗

bioRxiv2026

Highlighting strain rate dependent vibrational behavior of electrospun bundles for tendon/ligament and enthesis fascicle tissue regeneration

Enthesis tissue engineering aims to develop scaffolds that replicate the mechanical and structural gradients of the tendon/ligament-bone interface. Among the different biofabrication techniques, electrospinning is surely one of the most promising to fabricate morpho-mechanically relevant enthesis fascicle-inspired scaffolds. An interesting and totally unexplored characteristic of these nanofibrous scaffolds is their ability, when mechanically tested, to produce/transmit strain rate and nanofiber fracture-dependent mechanical vibrations, which can potentially influence surrounding tissues and cells. This study develops a method to investigate how scaffold geometry and material affect vibrational behavior under mechanical stimulation. Electrospun bundles of poly(L-lactic) acid/collagen type I (PLLA/Coll) were fabricated to mimic the fibrocartilage, the enthesis junction, and the tendon/ligament regions, while block copolymer poly(ethylene oxide terephthalate)-poly(butylene terephthalate) (PEOT-PBT) bundles represented only the tendon/ligament. Scaffolds were morphologically and mechanically characterized, including strain rate-dependent vibrational response. Scanning electron microscopy confirmed distinct fiber architectures. Under monotonic tensile tests to failure, scaffolds exhibited strain rate-dependent mechanical behavior, with PLLA/Coll bundles showing dominant vibrational frequencies up to 4.2 {+/-} 0.9 Hz with a scaffolds geometry-dependent manner. PEOT-PBT scaffolds instead, displayed higher vibration attenuation, with dominant frequencies peaking at 0.539 {+/-} 0.063 Hz. They also showed lower tensile properties, reflecting a different mechanical and vibrational profile respect to PLLA/Coll bundles. Integrating vibrational characterization with mechanical testing offers a novel framework for designing scaffolds that more accurately reproduce the gradient mechanical environment of fibrous musculoskeletal tissues such as tendons/ligaments and their entheses. These findings highlight the potential of this combined approach to further increase the mechanical comprehension of electrospun scaffolds. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/699441v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1e4f38dorg.highwire.dtl.DTLVardef@31e658org.highwire.dtl.DTLVardef@9c590org.highwire.dtl.DTLVardef@18996e9_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Wave-inspired MEW scaffolds for enhanced ligament tissue regeneration

AO_SCPLOWBSTRACTC_SCPLOWLigament injuries remain a major clinical challenge due to the limited intrinsic healing capacity of these fibrous tissues. Here, we demonstrate the use of melt electrowriting (MEW) to fabricate poly({varepsilon}-caprolactone) (PCL) scaffolds with precisely engineered wave architectures that mimic the hierarchical organization and nonlinear mechanics of native ligaments. By tuning fiber geometry, we achieved scaffolds with distinct mechanical behaviors ranging from highly compliant to structurally resilient, enabling architecture-driven modulation of elastic modulus and fatigue response. Mechanical testing revealed that wave-patterned scaffolds dissipate energy efficiently and adapt structurally under cyclic loading, reproducing key features of ligament-like viscoelasticity. When cultured with human anterior cruciate ligament (ACL) cells, the scaffolds supported adhesion, proliferation, and spatially organized alignment, together with the expression of ligament-associated markers. The results demonstrate that MEW scaffolds provide a favorable environment for ligament cell adhesion and matrix synthesis, while highlighting the strong influence of geometry on cell organization and early matrix production. Overall, this study establishes wave-based MEW architectures as a versatile platform to guide ligament tissue formation.

bioengineering↗

bioRxiv2025

Packed for Ossification: High-Density Bioprinting of hPDC Spheroids in HAMA for Endochondral Ossification

Long bone fractures are primarily repaired through endochondral ossification, a process in which a soft cartilage template forms at the injury site and is gradually replaced by bone. While bone has an innate self-healing capacity, this process can be disrupted in cases of large or complex defects, where regeneration fails, and clinical intervention is required. This study aimed at the development of a tissue engineering approach using human periosteum-derived cell (hPDC) spheroids encapsulated or bioprinted at high density within hyaluronic acid methacrylate (HAMA) hydrogels to support hypertrophic cartilage formation as a template for endochondral bone regeneration. We first compared different encapsulation time points (days 1, 7, and 14), finding that early encapsulation (day 1) enhanced spheroid fusion, increased DNA content, and promoted hypertrophic cartilage formation, as indicated by greater glycosaminoglycan (GAG) and collagen deposition along with lacunae formation. Next, HAMA-encapsulated spheroids were compared to spheroids formed using a standardized microwell platform, demonstrating that encapsulation promoted a more mature cartilage-like matrix with thicker collagen fibers and enhanced hypertrophic differentiation. Gene expression and immunostaining confirmed progression toward hypertrophic and osteogenic phenotypes. Finally, extrusion-based bioprinting of HAMA bioinks comprising a high-density of hPDC spheroids demonstrated scalability, improved spheroid alignment, and maintained robust cell viability and hypertrophic differentiation. HAs bioactivity and regulatory advantages support clinical translation, although achieving spatial control remains an area for further optimization. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/674866v2_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@40b55eorg.highwire.dtl.DTLVardef@434b2corg.highwire.dtl.DTLVardef@1fc4640org.highwire.dtl.DTLVardef@168252e_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

Hybrid bioinks for embedded bioprinting of an artery model

The integration of biomaterials with living cells and stimuli-responsive materials can be employed to create bioinks capable of generating 3D in vitro models that better recapitulate native tissues. We introduce a multilayered artery model that combines hybrid multifunctional materials including a stimuli-responsive polymeric ink to mimic the tunica adventitia arterial wall, and an extracellular matrix (ECM)-based bioink for the tunica media artery layer. The stimuli-responsive hybrid layer integrates inorganic (plasmonic nanoparticles) and organic (polymers) components, providing structural support and introducing diverse functionalities to the system. The cell-laden bioink consists of human vascular smooth muscle cells (vSMC) within a hydrogel based on porcine artery-derived decellularized extracellular matrix (dECM) that fosters optimal cell growth and proliferation. An embedding bioprinting technique was employed for the fabrication of the multimaterial artery model consisting of concentric cylinders. The dimensions of the 3D model and the bioprinting parameters were fine-tuned to ensure effective crosslinking of the multiple concentric layers resulting in the creation of self-supporting constructs. We demonstrate the effectiveness of the hybrid bioink composition and bioprinting parameters in supporting cell viability and proliferation within the multilayered construct, expanding the possibilities of employing novel multi-component materials for the fabrication of 3D vasculature models resembling the structure of native blood vessels.

bioengineering↗

bioRxiv2025

Dimethyl sulfoxide primes induced pluripotent stem cells for more efficient nephron progenitor and kidney organoid differentiation

The field of human induced pluripotent stem cells (hiPSCs) has seen significant progress since the discovery of reprogramming somatic cells using the transcription factors Oct4, Sox2, Klf4, and c-Myc. hiPSCs are similar to embryonic stem cells in a primed state of pluripotency and has the potential to differentiate into any adult human cell type, offering a versatile tool for research and potential therapeutic applications. However, the efficiency of differentiation protocols for generating complex structures with multiple cell types, like kidney organoids, remains a challenge. This study investigates the impact of treating hiPSCs with a low-dose dimethyl sulfoxide to enhance kidney organoid differentiation using a well-established protocol from literature. We found that treating hiPSCs with 1-2% DMSO affects gene expression of pluripotent transcription factors, hiPSC colony morphology, and enhances the expression of key metanephric mesenchyme nephron progenitor marker, SIX2 after 9 days of kidney organoid differentiation. Our findings also suggest that DMSO treatment helps improve hiPSC differentiation protocol efficiency toward the development of tubular kidney organoids. Further research is needed to elucidate the mechanisms underlying these effects and to refine the differentiation process for potential in vitro research applications in biomedical research and drug development.

cell biology↗

bioRxiv2024

Understanding the structure and mechanics of the sheep calcaneal enthesis: a relevant animal model for tissue engineering applications

Tendon/enthesis injuries are a worldwide clinical problem. Along the enthesis, collagen fibrils show a progressive loss of anisotropy and an increase in mineralization reaching the bone. This causes gradients of mechanical properties. The design of scaffolds to regenerate these load-bearing tissues requires of being validated in vivo in relevant large animal models. The sheep tendon of triceps surae muscle is an optimal animal model for this scope with limited knowledge about its structure and mechanics. We decided to understand in-depth its structure and full-field mechanics. Collagen fibrils morphology was investigated via scanning electron microscopy revealing a marked change in orientation/dimensions passing from tendon to enthesis. Backscatter electron images and nanoindentation at the enthesis/bone marked small gradients of mineralization at the mineralized fibrocartilage reaching 27%wt and indentation modulus around 17-30 GPa. The trabecular bone instead had indentation modulus around 15-22 GPa. Mechanical tensile tests with digital image correlation confirmed the typical non-linear behavior of tendons (failure strain = 8.2{+/-}1.0%; failure force = 1369{+/-}187 N) with maximum principal strains reaching mean values of {varepsilon}p1[~]7%. The typical auxetic behavior of tendon was highlighted by the minimum principal strains ({varepsilon}p2[~]5%), progressively dampened at the enthesis. Histology revealed that this behavior was caused by a local thickening of the epitenon. Cyclic tests showed a force loss of 21{+/-}7 % at the last cycle. These findings will be fundamental for biofabrication and clinicians interested in designing the new generation of scaffolds for enthesis regeneration. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/630234v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@143f0a8org.highwire.dtl.DTLVardef@16cb394org.highwire.dtl.DTLVardef@181be61org.highwire.dtl.DTLVardef@f9f2d9_HPS_FORMAT_FIGEXP M_FIG C_FIG Statement of SignificanceTendon and enthesis lesions are a clinical problem. To validate scaffolds for these applications large animal models are needed. Sheep tendon of triceps surae muscle is an optimal site for this scope. However, little is known about its extracellular matrix structure and mechanical properties. This work investigates the structure and mechanics of this tissue from different points of view. Scanning electron microscopy and histology studied its extracellular matrix morphology and composition. Backscattered electron images and nanoindentation assessed gradients of mineralization and stiffness at the enthesis. Mechanical tensile and cyclic tests coupled with digital image correlation elucidated its mechanics and superficial strain distribution. These findings will be fundamental for biofabrication and clinician experts to design innovative scaffolds to regenerate the enthesis.

bioengineering↗

bioRxiv2024

Revealing the Auxetic Behavior of Biomimetic Multi-material and Region-specific Nanofibrous Scaffolds via Synchrotron Multiscale Digital Volume Correlation: Innovative Building Blocks for the Enthesis Regeneration

Enthesis lesions are one of the prevalent causes of injuries in the tendon tissue. The gradient of mineralization, extracellular matrix organization and auxetic mechanical properties, make enthesis regeneration challenging. Innovative electrospun fascicle-inspired nanofibrous poly(L-lactic)acid/collagen type I blend scaffolds were developed. Specifically, a mineralized fibrocartilage-inspired region (with/without nano-mineralization with hydroxyapatite), where random and aligned nanofibers coexist, is connected to a tendon-like region made of aligned nanofibers, through a conical non-mineralized fibrocartilage-inspired junction. Scanning electron microscopy and synchrotron nano-tomography show the morphological biomimicry of scaffolds with the natural tendon fascicles. Human mesenchymal stromal cells spheroids cultures confirm a balanced expression of both tendon, cartilage and bone markers on the non-mineralized scaffolds compared with the mineralized ones. Mechanical tests, at different physiological strain-rates, reveal a biomimetic mechanical behavior of scaffolds and the ability of junctions to tune the mechanics of their surrounding sites. Multiscale synchrotron in situ tensile tests, coupled with Digital Volume Correlation, elucidate the full-field strain distribution of scaffolds from the structural down to the nanofiber level, highlighting the auxetic mechanical behavior of junctions typical of the natural enthesis. The findings and cutting-edge investigations of our study suggest the suitability of these enthesis-inspired fascicles as innovative scaffolds for enhanced enthesis regeneration.

bioengineering↗

bioRxiv2024

Embedding bioprinting of low viscous, photopolymerizable blood-based bioinks in a self-healing transparent supporting bath

Protein-based hydrogels have great potential to be used as bioinks for biofabrication-driven tissue regeneration strategies due to their innate bioactivity. Nevertheless, their use as bioinks in conventional 3D bioprinting is impaired due to their intrinsic low viscosity. Using embedding bioprinting, a liquid bioink is printed whithin a support that physically holds the patterned filament. Inspired by the recognized microencapsulation technique complex coacervation, we introduce crystal self-healing embedding bioprinting (CLADDING) based on a highly transparent crystal supporting bath. The suitability of distinct classes of gelatins was evaluated (i.e., molecular weight distribution, isoelectric point and ionic content), as well as the formation of gelatin-gum arabic microparticles as a function of pH, temperature, solvent and mass ratios. Characterizing and controlling this parametric window resulted in high yields of support bath with ideal self-healing properties for interaction with protein-based bioinks during bioprinting. This support bath achieved transparency, which boosted light permeation within the bath. CLADDING bioprinted constructs fully composed of platelet lysates encapsulating a co-culture of human mesenchymal stem cells and endothelial cells were obtained, demonstrating high-dense cellular network with excellent cell viability and stability over a month. CLADDING broadens the spectrum of photocrosslinkable materials with extremely low viscosity that can now be bioprinted with sensitive cells using embedding bioprinting without any additional support.

bioengineering↗

bioRxiv2023

RAVEN: development of a novel volumetric extrusion-based system for small-scale Additive Manufacturing

Recent technological advances in the field of Additive Manufacturing (AM) and the increasing need in Regenerative Medicine (RM) for devices that better and better mimic native tissues architecture are showing limitations in the current scaffolds fabrication techniques. A switch from the typical layer-by-layer approach is needed to achieve precise control on fibers orientation and pores dimension and morphology. In this work a new AM apparatus, the RAVEN (Robot-Assisted Volumetric ExtrusioN) system, is presented. RAVEN is based on a 7-DOF robotic arm and an FDM extruder and allows for volumetric extrusion of polymeric filaments. The development process, namely the robotic motion optimization, the optimization towards small-scale trajectories, the custom-made hardware/software interfaces, and the different printing capabilities are hereby presented. The successful results are promising towards future advanced applications such as in vivo bioprinting, in which the ability of the robot to change its configuration while printing will be crucial.

bioengineering↗

bioRxiv2022

Hypotrochoidal scaffolds for cartilage regeneration

The main function of articular cartilage is to provide a low friction surface and protect the underlying subchondral bone. The extracellular matrix composition of articular cartilage mainly consists of glycosaminoglycans and collagen type II. Specifically the collagen type II organization has a characteristic organization in three distinct zones; (1) the superficial zone which has collagen fibers oriented parallel to the surface, (2) the intermediate zone where there is no predominant orientation, and (3) the deep zone which shows a high orientation with fibers perpendicular to the underlying bone. Collagen type II fibers in these 3 zones take an arch-like organization that can be mimicked with segments of a hypotrochoidal curve. In this study, a script was developed that allowed the fabrication of scaffolds with a hypotrochoidal design. This design was investigated and compared to a regular 0-90 woodpile design. The results showed that the hypotrochoidal design was successfully fabricated. Micro-CT analyses divided the areas of the scaffold in their distinct zones. In addition, the mechanical analyses revealed that the hypotrochoidal design had a lower component Youngs modulus while the toughness and strain at yield were higher compared to the woodpile design. Fatigue tests showed that the hypotrochoidal design lost more energy per cycle due to the damping effect of the unique microarchitecture. Finite element analyses revealed that the hypotrochoidal design had an improved stress distribution compared to the 0-90 woodpile design due to the lower component stiffness. In addition, data from cell culture under dynamic stimulation demonstrated that the collagen type II deposition was improved in the hypotrochoidal design. Finally, Alcian blue staining revealed that the areas where the stress was higher during the stimulation produced more glycosaminoglycans. Our results highlight a new and simple scaffold design based on hypotrochoidal curves that could be used for cartilage tissue engineering.

bioengineering↗

bioRxiv2021

Soft, dynamic hydrogel confinement improves kidney organoid lumen morphology and reduces epithelial-mesenchymal transition in culture

Pluripotent stem cell-derived kidney organoids offer a promising solution to renal failure, yet current organoid protocols often lead to off-target cells and phenotypic alterations, preventing maturity. Here, we created various dynamic hydrogel architectures, conferring a controlled and biomimetic environment for organoid encapsulation. We investigated how hydrogel stiffness and stress relaxation affect renal phenotype and undesired fibrotic markers. We observed stiff hydrogel encapsulation led to an absence of certain renal cell types and signs of an epithelial- mesenchymal transition (EMT), whereas encapsulation in soft-stress-relaxing hydrogels led to all major renal segments, fewer fibrosis/EMT associated proteins, apical proximal tubule enrichment, and primary cilia formation, representing a significant improvement over current approaches to culture kidney organoids. Our findings show that engineering hydrogel mechanics and dynamics has a decided benefit for organoid culture. These structure-property- function relationships can enable rational design of materials, bringing us closer to functional engraftments and disease-modelling applications.

bioengineering↗