Search

This study proposes a tunable ink engineering methodology to allow 3D printing processability of highly bioactive but otherwise low-viscous and unprintable blood-derived materials. The hypothesis relies on improving the viscoelasticity and shear thinning behavior of platelet lysates (PL) and albumins (BSA) solutions by covalent coupling, enabling simultaneous extrusion and photocrosslinking upon filament deposition. The available amine groups on proteins (PL and BSA) are exploited for coupling with carboxyl groups present in methacrylated proteins (hPLMA and BSAMA), by leveraging carbodiimide chemistry. This reaction enabled the creation of a pre-gel from these extremely low-viscous materials (≈ 1 Pa), with precise tuning of the reaction, resulting in inks with a range of controlled viscosities and elasticities. Shape-fidelity analysis is performed on 3D-printed multilayered constructs, demonstrating the ability to reach clinically relevant sizes (>2 cm in size). After photocrosslinking, the scaffolds showcased a mechanically robust structure with sustained protein release over time. Bioactivity is evaluated using human adipose-derived stem cells, resulting in increased viability and metabolic activity over time. The herein described research methodology widens the possibilities for the use of low-viscosity materials in 3D printing but also enables the direct application of patient and blood-derived materials in precision medicine., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Synthetic hydrogels often lack the load-bearing capacity and mechanical properties of native biopolymers found in tissue, such as cartilage. In natural tissues, toughness is often imparted via the combination of fibrous noncovalent self-assembly with key covalent bond formation. This controlled combination of supramolecular and covalent interactions remains difficult to engineer, yet can provide a clear strategy for advanced biomaterials. Here, a synthetic supramolecular/covalent strategy is investigated for creating a tough hydrogel that embodies the hierarchical fibrous architecture of the extracellular matrix (ECM). A benzene-1,3,5-tricarboxamide (BTA) hydrogelator is developed with synthetically addressable norbornene handles that self-assembles to form a and viscoelastic hydrogel. Inspired by collagen's covalent cross-linking of fibrils, the mechanical properties are reinforced by covalent intra- and interfiber cross-links. At over 90% water, the hydrogels withstand up to 550% tensile strain, 90% compressive strain, and dissipated energy with recoverable hysteresis. The hydrogels are shear-thinning, can be 3D bioprinted with good shape fidelity, and can be toughened via covalent cross-linking. These materials enable the bioprinting of human mesenchymal stromal cell (hMSC) spheroids and subsequent differentiation into chondrogenic tissue. Collectively, these findings highlight the power of covalent reinforcement of supramolecular fibers, offering a strategy for the bottom-up design of dynamic, yet tough, hydrogels and bioinks., (© 2023 The Authors. Advanced Materials published by Wiley-VCH GmbH.)

Cartilage tissue presents low self-repair capability and lesions often undergo irreversible progression. Structures obtained by tissue engineering, such as those based in extrusion bioprinting of constructs loaded with stem cell spheroids may offer valuable alternatives for research and therapeutic purposes. Human mesenchymal stromal cell (hMSC) spheroids can be chondrogenically differentiated faster and more efficiently than single cells. This approach allows obtaining larger tissues in a rapid, controlled and reproducible way. However, it is challenging to control tissue architecture, construct stability, and cell viability during maturation. Herein, this work reports a reproducible bioprinting process followed by a successful post-bioprinting chondrogenic differentiation procedure using large quantities of hMSC spheroids encapsulated in a xanthan gum-alginate hydrogel. Multi-layered constructs are bioprinted, ionically crosslinked, and post chondrogenically differentiated for 28 days. The expression of glycosaminoglycan, collagen II and IV are observed. After 56 days in culture, the bioprinted constructs are still stable and show satisfactory cell metabolic activity with profuse extracellular matrix production. These results show a promising procedure to obtain 3D models for cartilage research and ultimately, an in vitro proof-of-concept of their potential use as stable chondral tissue implants., (© 2023 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.)

Traditional synthetic covalent hydrogels lack the native tissue dynamics and hierarchical fibrous structure found in the extracellular matrix (ECM). These dynamics and fibrous nanostructures are imperative in obtaining the correct cell/material interactions. Consequently, the challenge to engineer functional dynamics in a fibrous hydrogel and recapitulate native ECM properties remains a bottle-neck to biomimetic hydrogel environments. Here, the molecular tuning of a supramolecular benzene-1,3,5-tricarboxamide (BTA) hydrogelator via simple modulation of hydrophobic substituents is reported. This tuning results in fibrous hydrogels with accessible viscoelasticity over 5 orders of magnitude, while maintaining a constant equilibrium storage modulus. BTA hydrogelators are created with systematic variations in the number of hydrophobic carbon atoms, and this is observed to control the viscoelasticity and stress-relaxation timescales in a logarithmic fashion. Some of these BTA hydrogels are shear-thinning, self-healing, extrudable, and injectable, and can be 3D printed into multiple layers. These hydrogels show high cell viability for chondrocytes and human mesenchymal stem cells, establishing their use in tissue engineering applications. This simple molecular tuning by changing hydrophobicity (with just a few carbon atoms) provides precise control over the viscoelasticity and 3D printability in fibrillar hydrogels and can be ported onto other 1D self-assembling structures. The molecular control and design of hydrogel network dynamics can push the field of supramolecular chemistry toward the design of new ECM-mimicking hydrogelators for numerous cell-culture and tissue-engineering applications and give access toward highly biomimetic bioinks for bioprinting., (© 2023 The Authors. Advanced Materials published by Wiley-VCH GmbH.)

Spinal cord injuries result in severe neurological deficits and neuronal loss, with poor functional recovery. Mesenchymal stem cells have shown promising results; therefore the present objective of this work was to compare motor recovery after treatment with human dental pulp stem cells (hDPSC) cultivated in monolayer (2D) or as spheroids (3D), following avulsion and reimplantation of spinal motor roots in adult rats. Thus, 72 adult female Lewis rats were divided into 4 groups: avulsion (AV); avulsion followed by reimplantation (AR); avulsion associated with reimplant and 2D cell therapy (AR + 2D), and avulsion associated with reimplant and 3D cell therapy (AR + 3D). The application of the cells in 2D and 3D was performed by microsurgery, with subsequent functional assessment using a walking track test (Catwalk system), immunohistochemistry, neuronal survival, and qRT-PCR in 1-, 4-, and 12-weeks post-injury. The animals in the AR + 2D and AR + 3D groups showed the highest neuronal survival rates, and immunofluorescence revealed downregulation of GFAP, and Iba-1, with preservation of synaptophysin, indicating a reduction in glial reactivity, combined with the maintenance of pre-synaptic inputs. There was an increase in anti-inflammatory (IL-4, TGFβ) and a reduction of pro-inflammatory factors (IL-6, TNFα) in animals treated with reimplantation and hDPSC. As for the functional recovery, in all analyzed parameters, the AR + 2D group performed better and was superior to the avulsion alone. Overall, our results indicate that the 2D and 3D cell therapy approaches provide successful immunomodulation and motor recovery, consistent with advanced therapies after spinal cord injury., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Three-dimensional cellular aggregates can mimic the natural microenvironment of tissues and organs and obtaining them through controlled and reproducible processes is mandatory for scaling up and implementing drug cytotoxicity and efficacy tests, as well as tissue engineering protocols. The purpose of this work was to develop and evaluate the performance of a device with two different geometries fabricated by additive manufacturing. The methodology was based on casting a microwell array insert using a non-adhesive hydrogel to obtain highly regular microcavities to standardize spheroid formation and morphology. Spheroids of dental pulp stem cells, bone marrow stromal cells and embryonic stem cells showing high cell viability and average diameters of around 253, 220, and 500 μm, respectively, were produced using the device with the geometry considered most adequate. The cell aggregates showed sphericity indexes above 0.9 and regular surfaces (solidity index higher than 0.96). Around 1000 spheroids could be produced in a standard six-well plate. Overall, these results show that this method facilitates obtaining a large number of uniform, viable spheroids with pre-specified average diameters and through a low-cost and reproducible process for a myriad of applications., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Three-dimensional (3D) cell culture has tremendous advantages to closely mimic the in vivo architecture and microenvironment of healthy tissue and organs, as well as of solid tumors. Spheroids are currently the most attractive 3D model to produce uniform reproducible cell structures as well as a potential basis for engineering large tissues and complex organs. In this review we discuss, from an engineering perspective, processes to obtain uniform 3D cell spheroids, comparing dynamic and static cultures and considering aspects such as mass transfer and shear stress. In addition, computational and mathematical modeling of complex cell spheroid systems are discussed. The non-cell-adhesive hydrogel-based method and dynamic cell culture in bioreactors are focused in detail and the myriad of developed spheroid characterization techniques is presented. The main bottlenecks and weaknesses are discussed, especially regarding the analysis of morphological parameters, cell quantification and viability, gene expression profiles, metabolic behavior and high-content analysis. Finally, a vast set of applications of spheroids as tools for in vitro study model systems is examined, including drug screening, tissue formation, pathologies development, tissue engineering and biofabrication, 3D bioprinting and microfluidics, together with their use in high-throughput platforms., (© 2021 IOP Publishing Ltd.)

The transmembrane rabies virus glycoprotein (RVGP) is the main antigen of vaccine formulations used around the world to prevent rabies, the most lethal preventable infectious disease known. The objective of this work was to evaluate the potential of a bioreactor using wave-induced agitation in the initial steps of scaling up the rRVGP production process by a Drosophila melanogaster S2 cell line to produce rRVGP in sufficient quantities for immunization and characterization studies. Taking advantage of some remarkable features recognized in Drosophila S2 cells for scaling the culture process, a robust recombinant lineage (S2MtRVGPH-His) engineered by our group for the expression of rRVGP using a copper-inducible promoter was used in the bioreactor cultures. The WAVE Bioreactor was chosen because it represents an innovative approach to the cultivation of animal cells using single-use technology. For that purpose, we firstly established a procedure for culturing the S2MtRVGPH-His lineage in 100 mL Schott flasks. Using an inoculum of 5 × 10 5 cells/mL in culture medium (Sf900-III) induced with solution of CuSO 4 (0.7 mM) and a convenient pH range (6.2-7.0), optimal parameter values such as time of induction (72 h) and temperature (28 °C) to increase rRVGP production could be defined. This procedure was reproduced in culture experiments conducted in a WAVE Bioreactor™ 2/10 using a 2 L Cellbag. The results in Schott flasks and in WAVE Bioreactor™ were very similar, yielding a maximum titer of rRVGP above of 1 mg.L -1 . The immunization study showed that the rRVGP produced in the bioreactor was of high immunogenic quality.

Background: By and large, phototherapies are a promising approach to promote inactivation of microorganisms using light exposure, providing an effective alternative to control multidrug-resistant bacterial infections. Considering this, intercomparison between experiments is vitally important. Most experiments are performed using multiwell plates in which the volume of the culture medium is not standardized. In such cases, light attenuation and the distance it travels, which also depends on the volume and vessel geometry, can lead to different results. This study investigated how the different volumes imply different depths that light will have to travel through in this volume and the relation of this parameter with the result that will be obtained., Methods: Staphylococcus aureus was exposed to 460nm light with 50, 100 and 200J/cm 2 , in 100, 200, 500 and 1000μL of inocula in a 24-well plate to investigate pure light inactivation., Results: The literature suggests that fluence is the most important light parameter to obtain a high eradication of microbial cells in phototherapies. Our results show evidence that different geometrical configurations, taking into account the volume of the vessels, clearly affect the in vitro results, risking misinterpretation of dosimetry studies. Effects, such as dose distribution and decantation, are discussed throughout the paper., Conclusion: The outcome strongly depends on the volume and vessel geometry used. This study aims to encourage the standardization of phototherapies in vitro in general., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The effective treatment of antimicrobial modalities continues to be a serious challenge, mainly due to the increasing number of multidrug resistance pathogenic microorganisms. Microbial bioinhibition is an alternative method that has shown to be effective. This study investigated and described the effect of the visible light on five different microorganisms. The studied groups were composed by the species Acanthamoeba polyphaga, Candida albicans, Mycobacterium massiliense, Pseudomonas aeruginosa, and Staphylococcus aureus. These microorganisms were analyzed after six light doses exposition with three different wavelengths: 450, 520, and 630 nm. The present study indicates two different behaviors: bioinhibition and/or biostimulation. The bioinhibition effect was calculated using different percentages of the microorganism population, compared to the control group, in which the maximum value corresponds to 94% growth inhibition. The biostimulation effect was evaluated by the microorganism population increment for specific light doses. Our results showed a 132% population growth as the maximum value. These results were assessed by variance analysis. The Tukey's test was used for differentiating or comparing, depending on the circumstances. The obtained results suggested a visible light phototherapeutic effect that could be used as a microorganism inactivation method for the studied microorganisms. In some approaches, the biostimulation effect might also be a very interesting effect to be considered. This study supports the relevance of understanding the important role that phototherapy plays as a useful method for microbiological control studies and applications.