Biology subjects

Castilho, M.

Publications and source records attributed to Castilho, M..

8 recordsLinked to original sources

bioRxiv2026

Mechanoscribed Xolography for Multiscale Mechanical Patterning of Cell-laden Hydrogels

Mechanotransduction is a key regulator of healthy and malignant tissue development. However, current mechanobiological insights are largely derived from cells cultured on two dimensional substrates or in homogeneous biomaterials, which insufficiently capture the three dimensional (3D) heterogeneity and dynamic nature of the native extracellular matrix. This gap persists due to a lack of manufacturing processes and analysis modalities to control and monitor material properties and their evolution in 3D. Here, Mechanoscribed Xolography is introduced as a light sheet volumetric bioprinting process to rapidly fabricate 3D cell laden hydrogels with spatially controlled mechanical heterogeneity across multiple length scales. Moreover, synchronizing in-situ optical microscopy with printing enables autonomous detection and spatial location driven printing around cellular spheroids. The refinement and spatiotemporal evolution of material properties are assessed with Brillouin microscopy, which verifies that the contrast of imprinted heterogeneity is preserved over three weeks. Remarkably, regional differences in cell behavior are attained within a one-step, one-material construct with elasticity of approximately 0.3 kPa to 0.8 kPa, or 0.7 kPa to 2.9 kPa with a crosslinking-enhancing additive. This foundational work on high-precision, rapid bioprinting surrounding cellular objects represents a unique tool to systematically advance experimentation of 3D mechanotransduction and prospectively engineer cell instructive biomaterials for regenerative medicine.

bioengineering↗

bioRxiv2025

Rational Design and Modeling of Auxetic Fiber Scaffolds for Soft Tissue Engineering via Melt Electrowriting

BackgroundThe development of regenerative biomaterial scaffolds with mechanical properties tailored to native soft tissues remains a challenge in tissue engineering. Mechanical metamaterials with auxetic architectures have taken enormous flight over the past years, as they offer improved control over mechanical function and performance through spatially defined material architecture. However, despite their potential, auxetic scaffold designs are often developed empirically, and the relationship between scaffold microarchitecture, mechanical function, and manufacturability remains poorly defined, limiting their applicability in tissue engineering. In this study, we present a computational framework for the rational design of auxetic fiber scaffolds manufactured using Melt Electro-writing (MEW) for soft tissue engineering. To address the challenge of tailoring scaffold mechanical properties to native tissue, we integrated finite element method (FEM) modeling with experimental validation to predict the mechanical behavior of the scaffolds. MethodThe micro-fibrous scaffolds with customizable auxetic re-entrant structures were designed using Python scripts for generating G-code used in fabrication and for model definition in FEM modeling, respectively. Four distinct auxetic structures - HCELL, SREG, SINV, and STRI - were selected for this study to cover the wide range of mechanical properties of soft tissue, and their geometry parameters were adjusted to reduce the pore size while considering printing resolution. FEM modeling was employed to predict the mechanical behavior of the scaffolds. To validate the predictions, the scaffolds were manufactured via MEW and characterized mechanically under biaxial tensile loading. Results and conclusionMEW manufacturing enabled the production of auxetic fiber scaffolds with sub-millimeter pore resolution, while rational design allowed to correct fabrication discrepancies and improve printed design fidelity. Experimental biaxial characterization of the scaffolds revealed design-dependent, biphasic, non-linear mechanical response, and the developed FEM model was capable of not only accurately simulating the observed mechanical response but also accounting for variations introduced by MEW during fabrication. Additionally, auxetic scaffolds supported fibroblast viability over 7 days, confirming biocompatibility as a first step toward soft tissue engineering. The good alignment between numerical predictions and experimental results highlights the value of FEM modeling as a powerful computational tool for guiding the design and optimization of auxetic scaffolds with tailored mechanical performance for specific soft tissue requirements. The presented methodology offers a significant advancement in the development of functional biomaterials for biomedical applications.

bioengineering↗

bioRxiv2024

Xolography for Biomedical Applications: Dual-color Light-sheet Printing of Hydrogels with Local Control over Shape and Stiffness

AbstractCurrent challenges in tissue engineering include creation of extracellular environments that support and interact with cells using biochemical, mechanical, and structural cues. Spatial control over these cues is currently limited due to a lack of suitable fabrication techniques. This study introduces Xolography, an emerging dual-color light-sheet volumetric printing technology, to achieve control over structural and mechanical features for hydrogel-based photoresins at micro-to macroscale while printing within minutes. We propose a water-soluble photoswitch photoinitiator system and are the first to demonstrate Xolography with a library of naturally-derived, synthetic, and thermoresponsive hydrogels. Centimeter-scale, three-dimensional constructs with positive features of 20 {micro}m and negative features of [~] 100 {micro}m are fabricated with control over mechanical properties (compressive moduli 0.2 kPa - 6.5 MPa). Notably, switching from binary to grayscaled light projection enables spatial control over stiffness (0.2 - 16 kPa). As a proof of concept, grayscaled Xolography is leveraged with thermoresponsive hydrogels to introduce reversible anisotropic shape changes beyond isometric shrinkage. We finally demonstrate Xolography of viable cell aggregates, laying the foundation for cell-laden printing of dynamic, cell-instructive environments with tunable structural and mechanical cues in a fast one-step process. Overall, these innovations unlock unique possibilities of Xolography across multiple biomedical applications.

bioengineering↗

bioRxiv2023

Microstructured silk-fiber scaffolds with enhanced stretchability

Despite extensive research, current methods for creating three-dimensional (3D) silk fibroin (SF) scaffolds lack control over molecular rearrangement, particularly in the formation of {beta}-sheet nanocrystals, as well as hierarchical fiber organization at both micro- and macroscale. In this study, we introduce a fabrication process based on electrowriting of aqueous SF-based solutions followed by post-processing using an aqueous solution of sodium dihydrogen phosphate (NaH2PO4). This approach enables hierarchical assembly of SF chains via {beta}-sheet and -helix formation. Moreover, this process allows for precise control over micro- and macro-architectures in microfiber scaffolds, enabling the creation of 3D flat and tubular macrogeometries with square-based and crosshatch microarchitectures, featuring inter-fiber distances of 400 {micro}m and approximately 97% open porosity. Remarkably, the printed structures demonstrated restored {beta}-sheet and -helix structures, which imparted an elastic response of up to 20% deformation and the ability to support cyclic loading without plastic deformation. Furthermore, the printed constructs supported in vitro adherence and growth of human conditionally immortalized proximal tubular epithelial cells and glomerular endothelial cells, with cell viability above 95%. These cells formed uniform, aligned monolayers that deposited their own extracellular matrix. These findings represent a significant development in fabricating organized SF scaffolds with unique fiber structures, mechanical and biological properties, making them highly promising for regenerative medicine applications.

bioengineering↗

bioRxiv2023

3D printed magneto-active microfiber scaffolds for remote stimulation of 3D in vitro skeletal muscle models

Tunable culture platforms that guide cellular organization and mechanically stimulate skeletal muscle development are still unavailable due to limitations in biocompatibility and actuation triggered without contact. This study reports the rational design and fabrication of magneto-active microfiber meshes with controlled hexagonal microstructures via melt electrowriting (MEW) of a thermoplastic/graphene/iron oxide composite. In situ deposition of iron oxide nanoparticles on oxidized graphene yielded homogeneously dispersed magnetic particles with sizes above 0.5 m and low aspect ratio, preventing cellular internalization and toxicity. With these fillers, homogeneous magnetic composites with very high magnetic filler content (up to 10 wt.%) were obtained and successfully processed in a solvent-free manner for the first time. MEW of magnetic composites enabled the skeletal muscle-inspired design of hexagonal scaffolds with tunable fiber diameter, reconfigurable modularity, and zonal distribution of magneto-active and nonactive material. Importantly, the hexagonal microstructures displayed elastic deformability under tension, mitigating the mechanical limitations due to high filler content. External magnetic fields below 300 mT were sufficient to trigger out-of-plane reversible deformation leading to effective end-to-end length decrease up to 17%. Moreover, C2C12 myoblast culture on 3D Matrigel/collagen/MEW scaffolds showed that the presence of magnetic particles in the scaffolds did not significantly affect viability after 8 days with respect to scaffolds without magnetic filler. Importantly, in vitro culture demonstrated that myoblasts underwent differentiation at similar rates regardless of the presence of magnetic filler. Overall, these innovative microfiber scaffolds were proven as a magnetically deformable platform suitable for dynamic culture of skeletal muscle with potential for in vitro disease modeling.

bioengineering↗

bioRxiv2023

Matrix vesicle isolation from a three-dimensional in vitro bone model

Extracellular vesicles (EV) are nano-sized bilayer vesicles that are involved in biological functions and secreted by a wide variety of cells. Osteoblasts, the bone forming cells, can release a subset of EVs known as matrix vesicles (MtVs) which are believed to be involved in matrix mineralization and feature bone forming properties. Osteoblast-derived EVs or MtVs have been mostly isolated from conditions which are still far from nature, i.e. mesenchymal stromal cells (MSCs), or osteoblast cell lines cultured in two-dimensional (2D) tissue culture flasks. In our study, we aimed at investigating whether MtVs could also be isolated from an environment which better resembles the complex in vivo situation. This study investigated the EVs secretion during osteogenic differentiation of human bone marrow MSCs (hBMSCs) in the most advanced human three-dimensional (3D) in vitro woven bone constructs previously developed by our group. hBMSCs were cultured in spinner flask bioreactors which induced wall shear stress on cells and directed the cells to differentiate towards osteoblasts and osteocytes. The EVs secreted into the culture medium were isolated and characterized based on their morphological, biological, and functional properties. The characteristics of a part of isolated EVs shared similarities with MtVs. These vesicles were electron-dense and electron-lucent, showed alkaline phosphatase (ALP) activity, increased the amount of released free phosphate into the culture medium, and increased the amount of deposited phosphate within the ECM. The results indicate that a complex 3D environment mimicking bone development is favorable to stimulate MtV-producing cells to produce targeted MtVs in vitro. These MtVs potentially could be used as a biological agent for bone regeneration and fracture healing through, for instance, integration with biomaterials to target bone formation locally.

bioengineering↗

bioRxiv2020

Topographic Guidance in Melt-Electrowritten Tubular Scaffolds Enhances Engineered Kidney Tubule Performance

To advance the engineering of kidney tubules for future implantation, constructs should be both self-supportive and yet small-sized and highly porous. Here, we hypothesize that the fabrication of small-sized porous tubular scaffolds with a highly organized fibrous microstructure by means of melt-electrowriting (MEW) allows the development of self-supported kidney proximal tubules with enhanced properties. A custom-built MEW device was used to fabricate tubular fibrous scaffolds with small diameter sizes (O = 0.5, 1, 3 mm) and well-defined, porous microarchitectures (rhombus, square, and random). Human umbilical vein endothelial cells (HUVEC) and human conditionally immortalized proximal tubular epithelial cells (ciPTEC) were seeded into the scaffolds and tested for monolayer formation, integrity, and organization, as well as for extracellular matrix (ECM) production and renal transport functionality. Tubular scaffolds were successfully manufactured by fine control of MEW instrument parameters. A minimum inner diameter of 0.5 mm and pore sizes of 0.2 mm were achieved. CiPTEC formed tight monolayers in all scaffold microarchitectures tested, but well-defined rhombus-shaped pores outperformed and facilitated unidirectional cell orientation, increased collagen type IV deposition, and expression of the renal transporters and differentiation markers organic cation transporter 2 (OCT2) and P-glycoprotein (P-gp). To conclude, we present smaller diameter engineered kidney tubules with microgeometry-directed cell functionality. Due to the well-organized tubular fiber scaffold microstructure, the tubes are mechanically self-supported, and the self-produced ECM constitutes the only barrier between the inner and outer compartment, facilitating rapid and active solute transport.

bioengineering↗

bioRxiv2020

Impact of endotoxins in gelatine hydrogels on chondrogenic differentiation and inflammatory cytokine secretion in vitro

Gelatine methacryloyl (GelMA) hydrogels are widely used in studies aiming at cartilage regeneration. However, the endotoxin content of commercially available GelMAs and gelatines used in these studies is often overlooked, even though endotoxins may influence several cellular functions. Moreover, regulations for clinical use of biomaterials dictate a stringent endotoxin limit. We determined the endotoxin level of five different GelMAs and evaluated the effect on the chondrogenic differentiation of equine mesenchymal stromal cells (MSCs). Cartilage-like matrix production was evaluated by biochemical assays and immunohistochemistry. Furthermore, equine peripheral blood mononuclear cells (PBMCs) were cultured on the hydrogels for 24 hours, followed by the assessment of tumour necrosis factor (TNF)- and C-C motif chemokine ligand (CCL)2 as inflammatory markers. The GelMAs were found to have widely varying endotoxin content (two with >1000 EU/ml and three with <10 EU/ml), however, this was not a critical factor determining in vitro cartilage-like matrix production of embedded MSCs. PBMCs did produce significantly higher TNF- and CCL2 in response to the GelMA with the highest endotoxin level compared to the other GelMAs. Although limited effects on chondrogenic differentiation were found in this study, caution with the use of commercial hydrogels is warranted in the translation from in vitro to in vivo studies because of regulatory constraints and potential inflammatory effects of the content of these hydrogels.

bioengineering↗