3D bioprinted CRC model brings to light the replication necessity of an oncolytic vaccinia virus encoding FCU1 gene to exert an efficient anti-tumoral activity.

The oncolytic virus represents a promising therapeutic strategy involving the targeted replication of viruses to eliminate cancer cells, while preserving healthy ones. Despite ongoing clinical trials, this approach encounters significant challenges. This study delves into the interaction between an oncolytic virus and extracellular matrix mimics (ECM mimics). A three-dimensional colorectal cancer model, enriched with ECM mimics through bioprinting, was subjected to infection by an oncolytic virus derived from the vaccinia virus (oVV). The investigation revealed prolonged expression and sustained oVV production. However, the absence of a significant antitumor effect suggested that the virus's progression toward non-infected tumoral clusters was hindered by the ECM mimics. Effective elimination of tumoral cells was achieved by introducing an oVV expressing FCU1 (an enzyme converting the prodrug 5-FC into the chemotherapeutic compound 5-FU) alongside 5-FC. Notably, this efficacy was absent when using a non-replicative vaccinia virus expressing FCU1. Our findings underscore then the crucial role of oVV proliferation in a complex ECM mimics. Its proliferation facilitates payload expression and generates a bystander effect to eradicate tumors. Additionally, this study emphasizes the utility of 3D bioprinting for assessing ECM mimics impact on oVV and demonstrates how enhancing oVV capabilities allows overcoming these barriers. This showcases the potential of 3D bioprinting technology in designing purpose-fit models for such investigations.

Space radiation damage rescued by inhibition of key spaceflight associated miRNAs.

Our previous research revealed a key microRNA signature that is associated with spaceflight that can be used as a biomarker and to develop countermeasure treatments to mitigate the damage caused by space radiation. Here, we expand on this work to determine the biological factors rescued by the countermeasure treatment. We performed RNA-sequencing and transcriptomic analysis on 3D microvessel cell cultures exposed to simulated deep space radiation (0.5 Gy of Galactic Cosmic Radiation) with and without the antagonists to three microRNAs: miR-16-5p, miR-125b-5p, and let-7a-5p (i.e., antagomirs). Significant reduction of inflammation and DNA double strand breaks (DSBs) activity and rescue of mitochondria functions are observed after antagomir treatment. Using data from astronaut participants in the NASA Twin Study, Inspiration4, and JAXA missions, we reveal the genes and pathways implicated in the action of these antagomirs are altered in humans. Our findings indicate a countermeasure strategy that can potentially be utilized by astronauts in spaceflight missions to mitigate space radiation damage.

Confined bioprinting and culture in inflatable bioreactor for the sterile bioproduction of tissues and organs.

The future of organ and tissue biofabrication strongly relies on 3D bioprinting technologies. However, maintaining sterility remains a critical issue regardless of the technology used. This challenge becomes even more pronounced when the volume of bioprinted objects approaches organ dimensions. Here, we introduce a novel device called the Flexible Unique Generator Unit (FUGU), which is a unique combination of flexible silicone membranes and solid components made of stainless steel. Alternatively, the solid components can also be made of 3D printed medical-grade polycarbonate. The FUGU is designed to support micro-extrusion needle insertion and removal, internal volume adjustment, and fluid management. The FUGU was assessed in various environments, ranging from custom-built basic cartesian to sophisticated 6-axis robotic arm bioprinters, demonstrating its compatibility, flexibility, and universality across different bioprinting platforms. Sterility assays conducted under various infection scenarios highlight the FUGU's ability to physically protect the internal volume against contaminations, thereby ensuring the integrity of the bioprinted constructs. The FUGU also enabled bioprinting and cultivation of a 14.5 cm3 human colorectal cancer tissue model within a completely confined and sterile environment, while allowing for the exchange of gases with the external environment. This FUGU system represents a significant advancement in 3D bioprinting and biofabrication, paving the path toward the sterile production of implantable tissues and organs.

3D MODEL of an anatomically inert human hand: feasibility study.

OBJECTIVES: Surgery for congenital malformation of the hand is complex and protocols are not available. Simulation could help optimize results. The objective of the present study was to design, produce and assess a 3D-printed anatomical support, to improve success in rare and complex surgeries of the hand. MATERIAL AND METHODS: We acquired MRI imaging of the right hand of a 30 year-old subject, then analyzed and split the various skin layers for segmentation. Thus we created the prototype of a healthy hand, using 3D multi-material and silicone printing devices, and drew up a printing protocol suitable for all patients. We printed a base comprising bones, muscles and tendons, with a multi-material 3D printer, then used a 3D silicone printer for skin and subcutaneous fatty cell tissues in a glove-like shape. To evaluate the characteristics of the prototype, we performed a series of dissections on the synthetic hand and on a cadaveric hand in the anatomy lab, comparing realism, ease of handling and the final result of the two supports, and evaluated their respective advantages in surgical and training contexts. A grading form was given to each surgeon to establish a global score. RESULTS: This evaluation highlighted the positive and negative features of the model. The model avoided intrinsic problems of cadavers, such as muscle rigidity or tissue fragility and atrophy, and enables the anatomy of a specific patient to be rigorously respected. On the other hand, vascular and nervous networks, with their potential anatomical variants, are lacking. This preliminary phase highlighted the advantages and inconveniences of the prototype, to optimize the design and printing of future models. It is an indispensable prerequisite before performing studies in eligible pediatric patients with congenital hand malformation. CONCLUSION: The validation of 3D-printed anatomical model of a human hand opens a large field of applications in the area of preoperative surgical planning. The postoperative esthetic and functional benefit of such pre-intervention supports in complex surgery needs assessing.

Challenges in Nasal Cartilage Tissue Engineering to Restore the Shape and Function of the Nose.

The repair of nasal septal cartilage is a key challenge in cosmetic and functional surgery of the nose, as it determines its shape and its respiratory function. Supporting the dorsum of the nose is essential for both the prevention of nasal obstruction and the restoration of the nose structure. Most surgical procedures to repair or modify the nasal septum focus on restoring the external aspect of the nose by placing a graft under the skin, without considering respiratory concerns. Tissue engineering offers a more satisfactory approach, in which both the structural and biological roles of the nose are restored. To achieve this goal, nasal cartilage engineering research has led to the development of scaffolds capable of accommodating cartilaginous extracellular matrix-producing cells, possessing mechanical properties close to those of the nasal septum, and retaining their structure after implantation in vivo. The combination of a non-resorbable core structure with suitable mechanical properties and a biocompatible hydrogel loaded with autologous chondrocytes or mesenchymal stem cells is a promising strategy. However, the stability and immunotolerance of these implants are crucial parameters to be monitored over the long term after in vivo implantation, to definitively assess the success of nasal cartilage tissue engineering. Here, we review the tissue engineering methods to repair nasal cartilage, focusing on the type and mechanical characteristics of the biomaterials; cell and implantation strategy; and the outcome with regard to cartilage repair.

A Preliminary Study for an Intraoperative 3D Bioprinting Treatment of Severe Burn Injuries.

Intraoperative three-dimensional fabrication of living tissues could be the next biomedical revolution in patient treatment. APPROACH: We developed a surgery-ready robotic three-dimensional bioprinter and demonstrated that a bioprinting procedure using medical grade hydrogel could be performed using a 6-axis robotic arm in vivo for treating burn injuries. RESULTS: We conducted a pilot swine animal study on a deep third-degree severe burn model. We observed that the use of cell-laden bioink as treatment substantially affects skin regeneration, producing in situ fibroblast growth factor and vascular endothelial growth factor, necessary for tissue regeneration and re-epidermalization of the wound. CONCLUSIONS: We described an animal study of intraoperative three-dimensional bioprinting living tissue. This emerging technology brings the first proof of in vivo skin printing feasibility using a surgery-ready robotic arm-based bioprinter. Our positive outcome in skin regeneration, joined with this procedure's feasibility, allow us to envision the possibility of using this innovative approach in a human clinical trial in the near future.

From 3D printing to 3D bioprinting: the material properties of polymeric material and its derived bioink for achieving tissue specific architectures.

The application of 3D printing technologies fields for biological tissues, organs, and cells in the context of medical and biotechnology applications requires a significant amount of innovation in a narrow printability range. 3D bioprinting is one such way of addressing critical design challenges in tissue engineering. In a more general sense, 3D printing has become essential in customized implant designing, faithful reproduction of microenvironmental niches, sustainable development of implants, in the capacity to address issues of effective cellular integration, and long-term stability of the cellular constructs in tissue engineering. This review covers various aspects of 3D bioprinting, describes the current state-of-the-art solutions for all aforementioned critical issues, and includes various illustrative representations of technologies supporting the development of phases of 3D bioprinting. It also demonstrates several bio-inks and their properties crucial for being used for 3D printing applications. The review focus on bringing together different examples and current trends in tissue engineering applications, including bone, cartilage, muscles, neuron, skin, esophagus, trachea, tympanic membrane, cornea, blood vessel, immune system, and tumor models utilizing 3D printing technology and to provide an outlook of the future potentials and barriers.

Poloxamer/Poly(ethylene glycol) Self-Healing Hydrogel for High-Precision Freeform Reversible Embedding of Suspended Hydrogel.

Freeform reversible embedding of suspended hydrogel (FRESH) is an additive manufacturing technique enabling the 3D printing of soft materials with low or no yield stress. The printed material is embedded during the process until its solidification. From the literature, FRESH abilities are self-healing, reusability, suspending, thermal stability, and high-precision printing. This study proposes a new support hydrogel bath formulation for FRESH 3D printing. To do so, a poloxamer micellar thermoreversible hydrogel is tuned through the addition of poly(ethylene glycol) (PEG) to adapt rheological properties. PEG macromolecules interact with poly(ethylene oxide) blocks of poloxamer and favor micelle dehydration, and then decreasing the gelation temperature, the yield stress, and the viscosity. Parameters such as the Oldroyd number and the Rayleigh-Plateau instability, both dependent on yield stress, were studied to determine their impact on the FRESH 3D printing resolution and accuracy. It was found that print accuracy of embedded parts increases with increasing yield stress but then the self-healing property gets limited, leading to crevasse formation. The usefulness of this approach is distinctly demonstrated through a six-axis printing of a highly complex silicone anatomical model. Printing fidelity of 96.0 ± 3.58% (5-40 mm printed parts) is thus achieved using the newly formulated FRESH material, while only 56.0 ± 0.76% fidelity is obtained using the standard formulation. The present study thus showed that complex FRESH 3D printing of soft materials is possible in this tunable hydrogel and that parts can be manufactured on an industrial scale, thanks to the reusability of the support bath.

Tissue engineering of retina through high resolution 3-dimensional inkjet bioprinting.

The mammalian retina contains multiple cellular layers, each carrying out a specific task. Such a controlled organization should be considered as a crucial factor for designing retinal therapies. The maintenance of retinal layered complexity through the use of scaffold-free techniques has recently emerged as a promising approach for clinical ocular tissue engineering. In an attempt to fabricate such layered retinal model, we are proposing herein a unique inkjet bioprinting system applied to the deposition of a photoreceptor cells (PRs) layer on top of a bioprinted retinal pigment epithelium (RPE), in a precise arrangement and without any carrier material. The results showed that, after bioprinting, both RPE and PRs were well positioned in a layered structure and expressed their structural markers, which was further demonstrated by ZO1, MITF, rhodopsin, opsin B, opsin R/G and PNA immunostaining, three days after bioprinting. We also showed that considerable amounts of human vascular endothelial growth factors (hVEGF) were released from the RPE printed layer, which confirmed the formation of a functional RPE monolayer after bioprinting. Microstructures of bioprinted cells as well as phagocytosis of photoreceptor outer segments by apical RPE microvilli were finally established through transmission electron microscopy (TEM) imaging. In summary, using this carrier-free bioprinting method, it was possible to develop a reasonable in vitro retina model for studying some sight-threatening diseases, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP).

Multifunctional polymeric implant coatings based on gelatin, hyaluronic acid derivative and chain length-controlled poly(arginine).

Surface of the implantable devices is the root cause of several complications such as infections, implant loosening and chronic inflammation. There is an urgent need for multifunctional coatings that can address these shortcomings simultaneously in a manner similar to the structures of extracellular matrix. Herein, we developed a coating system composed of ECM components and a naturally derived polypeptide. The interactions between the coating components create an environment that enables incorporation of an antimicrobial/angiogenic polypeptide. The film composition is based gelatin and hyaluronic acid modified with aldehyde groups (HA-Ald) that can react with poly (arginine) (PAR) through transient interactions. Nanoplasmon measurements demonstrated a significantly higher loading of PAR in films containing HA-Ald with longer retention of PAR in the structure. The presence of PAR not only provides to the film surface antimicrobial (contact-killing) properties but also increased endothelial cell-cell contacts (PECAM) and VEGFA gene expression and secretion by human vascular endothelial cells. This multifunctional coating can be easily applied to surface of implants where it can enact on several problems simultaneously.

Development of an electrochemical detection system for measuring DNA methylation levels using methyl CpG-binding protein and glucose dehydrogenase-fused zinc finger protein.

DNA methylation level at a certain gene region is considered as a new type of biomarker for diagnosis and its miniaturized and rapid detection system is required for diagnosis. Here we have developed a simple electrochemical detection system for DNA methylation using methyl CpG-binding domain (MBD) and a glucose dehydrogenase (GDH)-fused zinc finger protein. This analytical system consists of three steps: (1) methylated DNA collection by MBD, (2) PCR amplification of a target genomic region among collected methylated DNA, and (3) electrochemical detection of the PCR products using a GDH-fused zinc finger protein. With this system, we have successfully measured the methylation levels at the promoter region of the androgen receptor gene in 106 copies of genomic DNA extracted from PC3 and TSU-PR1 cancer cell lines. Since no sequence analysis or enzymatic digestion is required for this detection system, DNA methylation levels can be measured within 3h with a simple procedure.

Multiplatform Biomarker Discovery for Bladder Cancer Recurrence Diagnosis.

Purpose. Nonmuscle invasive bladder cancer (BCa) has a high recurrence rate requiring lifelong surveillance. Urinary biomarkers are promising as simple alternatives to cystoscopy for the diagnosis of recurrent bladder cancer. However, no single marker can achieve the required accuracy. The purpose of this study was to select a multiparameter panel, comprising urinary biomarkers and clinical parameters, for BCa recurrence diagnosis. Experimental Design. Candidate biomarkers were measured in urine samples of BCa patients with recurrence and BCa patients without recurrence. A multiplatform strategy was used for marker quantification comprising a multiplexed microarray and an automated platform for ELISA analysis. A multivariate statistical analysis combined the results from both platforms with the collected clinical data. Results. The best performing combination of biomarkers and clinical parameters achieved an AUC value of 0.91, showing better performance than individual parameters. This panel comprises six biomarkers (cadherin-1, IL-8, ErbB2, IL-6, EN2, and VEGF-A) and three clinical parameters (number of past recurrences, number of BCG therapies, and stage at time of diagnosis). Conclusions. The multiparameter panel could be a useful noninvasive tool for BCa surveillance and potentially impact the clinical management of this disease. Validation of results in an independent cohort is warranted.

Cancer-Cells on Chip for Label-Free Detection of Secreted Molecules.

In the present report, we are making the proof of concept of cell small populations (from 1 to 100 cells) spotting, culture and secretion detection on a gold surface. In order to keep the cells in a hydrated environment during the robotized micropipetting and to address different cell lines on a single chip, a biocompatible alginate polymer was used. This approach enables the encapsulation of the cell in a very small volume (30 nL), directly on the substrate and permits a precise control of the number of cells in each alginate bead. After 24 h of culture, the adherent cells are ready for surface plasmon resonance imaging (SPRi) experimentation. To enable the detection of secreted proteins, various antibodies are immobilized in an organized manner on a SPRi sensor and permitted the multiplex detection of different proteins secreted by the different cultured cell lines. Evidence of the real-time detection will be presented for Prostate Specific Antigen (PSA) and ß-2-microglobulin (B2M) secreted by prostate cancer cells following induction by dihydrotestosterone (DHT). Different kinetics for the two secreted proteins were then demonstrated and precisely determined using the chip.

Disease map-based biomarker selection and pre-validation for bladder cancer diagnostic.

CONTEXT: Urinary biomarkers are promising as simple alternatives to cystoscopy for the diagnosis of de novo and recurrent bladder cancer. OBJECTIVE: To identify a highly sensitive and specific biomarker candidate set with potential clinical utility in bladder cancer. MATERIALS AND METHODS: Urinary biomarker concentrations were determined by ELISA. The performance of individual markers and marker combinations was assessed using ROC analysis. RESULTS: A five-biomarker panel (IL8, MMP9, VEGFA, PTGS2 and EN2) was defined from the candidate set. DISCUSSION AND CONCLUSION: This panel showed a better overall performance than the best individual marker. Further validation studies are needed to evaluate its clinical utility in bladder cancer.

Polymer adhesive surface as flexible generic platform for multiplexed assays biochip production.

The present report describes the integration and application possibilities of a new microarray concept based on adhesive surface. The method was shown to enable the straightforward production of 384 and 1536-well plates modified with 100 and 25 spots per well, respectively. Such in-well densities were only possible thanks to the fabrication process which implies first the deposition of the microarray on a flat adhesive surface and then its assembly with bottomless 384 or 1536-well plates. The concept was also confronted to various applications such as oligonucleotide detection, localised cell culture onto spotted adhesion proteins and immobilisation of peptide or active antibodies for immunoassays. In the particular case of immunotesting, the study focused on liver diseases diagnosis and more particularly on the detection of either one liver cancer marker, the alpha-fetoprotein, or the detection of Hepatitis C Virus infection. In every cases, interesting performances were obtained directly in crude patient serum, proof of the robust and generic aspect of the platform.

Protein-diazonium adduct direct electrografting onto SPRi-biochip.

A direct protein immobilization method for surface plasmon resonance imaging (SPRi) gold chip arraying is exposed. The biomolecule electroaddressing strategy, previously demonstrated by our team on carbon surfaces, is here valuably involved and adapted to create a straightforward and efficient protein immobilization process onto SPRi-biochips. The proteins, modified with an aryl-diazonium adduct, are addressed to the SPRi chip surface through the electroreduction of the aryl-diazonium. The biomolecule deposition was followed through SPRi live measurements during the electrografting process. A specially designed setup enabled us to directly observe the mass increasing at the sensor surface while the proteins were electrografted. A pin electrospotting method, allowing the achievement of distinct sensing layers on gold SPRi-biochips, was used to generate microarray biochips. The integrity of the immobilized proteins and the specificity of the detection, based on antigen/antibody interactions, were demonstrated for the detection of specific antibodies and ovalbumin. The SPRi detection limit of ovalbumin using the electroaddressing of anti-ovalbumin IgG was compared with two other immobilization procedures, cystamine-glutaraldehyde self-assembled monolayer and pyrrole, and was found to be a decade lower than these ones (100 ng/mL, i.e., 2 nM).

Cylinder-shaped conducting polypyrrole for labelless electrochemical multidetection of DNA.

A new multidetection biosensor has been developed using the electrochemical properties of cylinder-shaped conducting polypyrrole grown on miniaturized graphite electrodes. Our objective was to conceive a sensitive, labelless and real-time DNA sensor for biomedical diagnosis. In a first step, copolymers bearing both ferrocene redox markers and oligonucleotide probes were selectively electro-addressed on microchip electrodes. Then, the study of their voltammetric response upon the addition of DNA targets revealed that the hybridization was efficiently transduced through the variation of ferrocene oxidation intensity. Using this technique, a good selectivity between Human Immunodeficiency Virus and Hepatitis B Virus targets was obtained. It was indeed possible to directly follow the hybridization. Complementary DNA detection limit reached 100 pM (3 fmol in 30 microL), which represents a good performance for such a practical, labelless and real-time sensor.

On-chip chemiluminescent signal enhancement using nanostructured gold-modified carbon microarrays.

An original method for the enhancement of chemiluminescent (CL) on-chip detection of protein and oligonucleotides is presented. This enhancement is based on the electrodeposition of a gold nanostructured layer onto a screen-printed (SP) carbon microarray prior to the immobilization of biomolecules through a well-established diazonium adduct electrodeposition. Morphological studies of the Au layer (optical and atomic force microscopy) show that the metal film is composed of nanostructured 800 nm diameter particles covering the entire graphite surface and yielding a high surface area. Using these modified SP microarrays, enhancement factors of 229 and 126 were obtained for prostate-specific antigen (PSA) and p53 oligonucleotide detection, respectively. These enhancements were associated with three different phenomena: an enhancement of the catalyzed chemiluminescent reaction by the gold surface, an increase of the specific surface area for immobilization of the probe biomolecules, and an opposite quenching effect due to the overlapping of the gold absorption and CL emission peaks. For free PSA and target oligonucleotide detection, enhanced performances were obtained, giving detection limits of 5 ng/mL and 0.1 nM, respectively.

Electroaddressed immobilization of recombinant HIV-1 P24 capsid protein onto screen-printed arrays for serological testing.

A serological chemiluminescent biochip was designed based on screen-printed electrode arrays composed of nine 1-mm(2) electrodes. Arrays were shown to be produced with good batch-to-batch reproducibility (standard deviations of 4.4 and 12.0% for ferricyanide oxidation potential and current, respectively) and very good reproducibility within a particular array (2.0 and 7.5% standard deviations for the same controls). Electrode arrays were used to electroaddress various bioconjugate structures comprising a recombinant HIV-1 P24 capsid protein (RH24K) in polypyrrole film. Entrapment of RH24K preimmobilized onto maleic anhydride-alt-methyl vinyl ether copolymer was shown to be the more efficient immobilization procedure. This addressed sensing layer enabled the detection of anti-P24 antibodies at a concentration of 3.5 ng/ml through peroxidase-labeled anti-human immunoglobulin G reaction. The biochip was used to perform an HIV-1 serological test in human sera. HIV-1 seropositive and seronegative sera were easily discriminated using serum dilutions greater than 1/10,000.