Three-Dimensionally Printed Biphasic Calcium Phosphate Ceramic Substrates as the Sole Inducer of Osteogenic Differentiation in Stromal Vascular Fraction Cells.

The stromal vascular fraction (SVF) is a derivate of fat tissue comprising both adipose-derived mesenchymal stem cells and endothelial cells and serves as a promising cell source for engineering vascularized bone tissues. Its combination with osteoconductive biphasic calcium phosphate (BCP) ceramic may represent a point-of-care agent for bone reconstruction. Here we assessed the proliferation and osteogenic differentiation capacities of SVF on 3D printed BCP implants, in comparison with isolated adipose-derived mesenchymal stem cells (AD-MSCs). AD-MSCs and SVF isolated from human donors were seeded on plastic or 3D printed BCP ceramics with sinusoidal or gyroid macrotopography and cultured in the presence or absence of osteogenic factors. Vascular, hematopoietic and MSC surface markers were assessed by flow cytometry whereas osteogenic activity was investigated through alizarin red staining and alkaline phosphatase activity. Osteogenic factors were necessary to trigger osteogenic activity when cells were cultured on plastic, without significant difference observed between the two cell populations. Interestingly, osteogenic activity was observed on BCP implants in the absence of differentiation factors, without significant difference in level activity between the two cell populations and macrotopography. This study offers supportive data for the use of combined BCP scaffolds with SVF in a perspective of a one-step surgical procedure for bone regeneration.

Tuning viscoelasticity and stiffness in bioprinted hydrogels for enhanced 3D cell culture: A multi-scale mechanical analysis.

Bioprinted hydrogels are extensively studied to provide an artificial matrix for 3D cell culture. The success of bioprinting hydrogels relies on fine-tuning their rheology and composition to achieve shear-thinning behavior. However, a challenge arises from the limited viscoelastic and stiffness range accessible from a single hydrogel formulation. Nevertheless, hydrogel mechanical properties are recognized as essential cues influencing cell phenotype, migration, and differentiation. Thus, it is crucial to develop a system to easily modulate bioprinted hydrogels' mechanical behaviors. In this work, we modulated the viscoelastic properties and stiffness of bioprinted hydrogels composed of fibrinogen, alginate, and gelatin by tuning the crosslinking bath solution. Various concentrations of calcium ionically crosslinked alginate, while transglutaminase crosslinked gelatin. Subsequently, we characterized the mechanical behavior of our bioprinted hydrogels from the nanoscale to the macroscale. This approach enabled the production of diverse bioprinted constructs, either with similar elastic behavior but different elastic moduli or with similar elastic moduli but different viscoelastic behavior from the same hydrogel formulation. Culturing fibroblasts in the hydrogels for 33 days revealed a preference for cell growth and matrix secretion in the viscoelastic hydrogels. This work demonstrates the suitability of the method to decouple the effects of material mechanical from biochemical composition cues on 3D cultured cells.

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.

Targeting the Complexity of In Vitro Skin Models: A Review of Cutting-Edge Developments.

Skin in vitro models offer much promise for research, testing drugs, cosmetics, and medical devices, reducing animal testing and extensive clinical trials. There are several in vitro approaches to mimicking human skin behavior, ranging from simple cell monolayer to complex organotypic and bioengineered 3-dimensional models. Some have been approved for preclinical studies in cosmetics, pharmaceuticals, and chemicals. However, development of physiologically reliable in vitro human skin models remains in its infancy. This review reports on advances in in vitro complex skin models to study skin homeostasis, aging, and skin disease.

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.

Increasing Collagen to Bioink Drives Mesenchymal Stromal Cells-Chondrogenesis from Hyaline to Calcified Layers.

The bioextrusion of mesenchymal stromal cells (MSCs) directly seeded in a bioink enables the production of three-dimensional (3D) constructs, promoting their chondrogenic differentiation. Our study aimed to evaluate the effect of different type I collagen concentrations in the bioink on MSCs' chondrogenic differentiation. We printed 3D constructs using an alginate, gelatin, and fibrinogen-based bioink cellularized with MSCs, with four different quantities of type I collagen addition (0.0, 0.5, 1.0, and 5.0 mg per bioink syringe). We assessed the influence of the bioprinting process, the bioink composition, and the growth factor (TGF-ꞵ1) on the MSCs' survival rate. We confirmed the biocompatibility of the process and the bioinks' cytocompatibility. We evaluated the chondrogenic effects of TGF-ꞵ1 and collagen addition on the MSCs' chondrogenic properties through macroscopic observation, shrinking ratio, reverse transcription polymerase chain reaction, glycosaminoglycan synthesis, histology, and type II collagen immunohistochemistry. The bioink containing 0.5 mg of collagen produces the richest hyaline-like extracellular matrix, presenting itself as a promising tool to recreate the superficial layer of hyaline cartilage. The bioink containing 5.0 mg of collagen enhances the synthesis of a calcified matrix, making it a good candidate for mimicking the calcified cartilaginous layer. Type I collagen thus allows the dose-dependent design of specific hyaline cartilage layers.

In situ transient transfection of 3D cell cultures and tissues, a promising tool for tissue engineering and gene therapy.

Various research fields use the transfection of mammalian cells with genetic material to induce the expression of a target transgene or gene silencing. It is a tool widely used in biological research, bioproduction, and therapy. Current transfection protocols are usually performed on 2D adherent cells or suspension cultures. The important rise of new gene therapies and regenerative medicine in the last decade raises the need for new tools to empower the in situ transfection of tissues and 3D cell cultures. This review will present novel in situ transfection methods based on a chemical or physical non-viral transfection of cells in tissues and 3D cultures, discuss the advantages and remaining gaps, and propose future developments and applications.

3D Bioprinting in Microgravity: Opportunities, Challenges, and Possible Applications in Space.

3D bioprinting has developed tremendously in the last couple of years and enables the fabrication of simple, as well as complex, tissue models. The international space agencies have recognized the unique opportunities of these technologies for manufacturing cell and tissue models for basic research in space, in particular for investigating the effects of microgravity and cosmic radiation on different types of human tissues. In addition, bioprinting is capable of producing clinically applicable tissue grafts, and its implementation in space therefore can support the autonomous medical treatment options for astronauts in future long term and far-distant space missions. The article discusses opportunities but also challenges of operating different types of bioprinters under space conditions, mainly in microgravity. While some process steps, most of which involving the handling of liquids, are challenging under microgravity, this environment can help overcome problems such as cell sedimentation in low viscous bioinks. Hopefully, this publication will motivate more researchers to engage in the topic, with publicly available bioprinting opportunities becoming available at the International Space Station (ISS) in the imminent future.

Quantification of cell contractile behavior based on non-destructive macroscopic measurement of tension forces on bioprinted hydrogel.

Contraction assay based on surface measurement have been widely used to evaluate cell contractility in 3D models. This method is straightforward and requires no specific equipment, but it does not provide quantitative data about contraction forces generated by cells. We expanded this method with a new biomechanical model, based on the work-energy theorem, to provide non-destructive longitudinal monitoring of contraction forces generated by cells in 3D. We applied this method on hydrogels seeded with either fibroblasts or osteoblasts. Hydrogel mechanical characteristics were modulated to enhance (condition HCAHigh: hydrogel contraction assay high contraction) or limit (condition HCALow: hydrogel contraction assay low contraction) cell contractile behaviors. Macroscopic measures were further correlated with cell contractile behavior and descriptive analysis of their physiology in response to different mechanical environments. Fibroblasts and osteoblasts contracted their matrix up to 47% and 77% respectively. Contraction stress peaked at day 5 with 1.1 10-14 Pa for fibroblasts and 3.5 10-14 Pa for osteoblasts, which correlated with cell attachment and spreading. Negligible contraction was seen in HCALow. Both fibroblasts and osteoblasts expressed α-SMA contractile fibers in HCAHigh and HCALow. Failure to contract HCALow was attributed to increased cross-linking and resistance to proteolytic degradation of the hydrogel.

Passive sampling of environmental DNA in aquatic environments using 3D-printed hydroxyapatite samplers.

The study of environmental DNA (eDNA) released by aquatic organisms in their habitat offers a fast, noninvasive and sensitive approach to monitor their presence. Common eDNA sampling methods such as water filtration and DNA precipitation are time-consuming, require difficult-to-handle equipment and partially integrate eDNA signals. To overcome these limitations, we created the first proof of concept of a passive, 3D-printed and easy-to-use eDNA sampler. We designed the samplers from hydroxyapatite (HAp samplers), a natural mineral with a high DNA adsorption capacity. The porous structure and shape of the samplers were designed to optimize DNA adsorption and facilitate their handling in the laboratory and in the field. Here we show that HAp samplers can efficiently collect genomic DNA in controlled set-ups, but can also collect animal eDNA under controlled and natural conditions with yields similar to conventional methods. However, we also observed large variations in the amount of DNA collected even under controlled conditions. A better understanding of the DNA-hydroxyapatite interactions on the surface of the samplers is now necessary to optimize eDNA adsorption and to allow the development of a reliable, easy-to-use and reusable eDNA sampling tool.

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.

Vascular Tissue Engineering: Challenges and Requirements for an Ideal Large Scale Blood Vessel.

Since the emergence of regenerative medicine and tissue engineering more than half a century ago, one obstacle has persisted: the in vitro creation of large-scale vascular tissue (>1 cm3) to meet the clinical needs of viable tissue grafts but also for biological research applications. Considerable advancements in biofabrication have been made since Weinberg and Bell, in 1986, created the first blood vessel from collagen, endothelial cells, smooth muscle cells and fibroblasts. The synergistic combination of advances in fabrication methods, availability of cell source, biomaterials formulation and vascular tissue development, promises new strategies for the creation of autologous blood vessels, recapitulating biological functions, structural functions, but also the mechanical functions of a native blood vessel. In this review, the main technological advancements in bio-fabrication are discussed with a particular highlights on 3D bioprinting technologies. The choice of the main biomaterials and cell sources, the use of dynamic maturation systems such as bioreactors and the associated clinical trials will be detailed. The remaining challenges in this complex engineering field will finally be discussed.

Biomechanical and functional comparison of moulded and 3D printed medical silicones.

Modern 3D printing of implantable devices provides an important opportunity for the development of personalized implants with good anatomical fit. Nevertheless, 3D printing of silicone has been challenging and the recent advances in technology are provided by the systems which can print medical grade silicone via extrusion. However, the potential impacts of the 3D printing process of silicone on its biomechanical properties has not been studied in sufficient detail. Therefore, the present study compares 3D printed and moulded silicone structures for their cytotoxicity, surface roughness, biomechanical properties, and in vivo tissue reaction. The 3D printing process creates increased nanoscale roughness and noticeably changes microscale topography. Neither the presence of these features nor the differences in processes were found to result in an increase in cytotoxicity or tissue reaction for 3D printed structures, exhibiting limited inflammatory reaction and cell viability above the threshold values. On the contrary, the biomechanical properties have demonstrated significant differences in static and dynamic conditions, and in thermal expansion. Our results demonstrate that 3D printing can be used for establishing a better biomechanical microenvironment for the surrounding tissue of the implant particularly for fragile soft tissue like epithelial mucosa without having any negative effect on the cytotoxicity or in vivo reaction to silicone. For engineering of the implants, however, one must consider the differences in mechanical properties to result in correct and personalized geometry and proper physical interaction with tissues.

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.

Gold nanobipyramids integrated ultrasensitive optical and electrochemical biosensor for Aflatoxin B1 detection.

This work reports the development of an electrical and optical biosensing for label-free detection of Aflatoxin B1 (AFB1) using gold (Au) nanobipyramids (NBPs). AuNBPs were synthesized through a two-step seed-mediated growth process followed by an exchange of capping agent from surfactant to lipoic acid. Pure and monodispersed AuNBPs of 70 nm base length were obtained and deposited on indium tin oxide (ITO)-coated glass substrate modified with self-assembled (3-Aminopropyl) triethoxysilane (APTES) film. The characterization of the obtained surfaces using spectroscopy, microscopy and diffractometry confirms the formation of AuNBPs, the conjugation to ITO electrode substrate and the immobilization of anti-AFB1 antibodies. AuNBPs modified ITO substrates were used for both electrochemical and Surface Plasmon Resonance biosensing studies. Localized Surface Plasmon Resonance (LSPR) local field enhancement was demonstrated. SPR based AFB1 detection was found to be linear in the 0.1-500 nM range with a limit of detection of 0.4 nM, whereas, impedimetric AFB1 detection was shown to be linear in the 0.1-25 nM range with a limit of detection of 0.1 nM. The practical utility of the impedimetric sensor was tested in spiked maize samples and 95-100% recovery percentage was found together with low relative standard deviation, proof of the robustness of this AFB1 sensor.