Tissue Printing Research Articles

3D bioprinting meniscus tissue onboard the International Space Station

We bioprinted meniscus tissue on the International Space Station (ISS) using the onboard BioFabrication Facility (BFF). The three dimensional (3D) printing bioink, cells, culture media and fixative were delivered to the ISS on NG-18 and SpX-27 vehicles and stored prior to the printing operation. The meniscus tissue was fabricated from ink composed of collagens type I and II, chondroitin sulfate and mesenchymal stem cells. Following printing, the meniscus tissue was cultured for 2 weeks in growth media, then stored at 4 °C and returned to earth for analysis. The print showed good overall shape fidelity, and dimensions were comparable to control meniscus tissue printed on Earth. Young's modulus of the ISS printed meniscus was approximately 4-fold lower than the control. Histologic evaluation showed good cell distribution within the print. Though logistical challenges were encountered during payload delivery to the ISS and operational challenges limited the cell culture portion of this study, this investigation demonstrated the feasibility for 3D printed musculoskeletal tissue in microgravity. The completed meniscus tissue print is the largest tissue engineered model 3D printed on the ISS to date, the first to be 3D bioprinted using an ink similar in composition to native tissue, and the first to be fabricated on the ISS in an anatomically relevant shape. These experiments help advance the field of tissue engineering in low or microgravity where 3D bioprinting may have a role in future long term space flight or extraterrestrial habitation.

Extracellular matrix decellularization approaches for 3D tissue printing

3D bioprinting holds transformative potential for the field of regenerative medicine, offering unprecedented opportunities for the fabrication of complex, living tissues. Central to this technological innovation is the development of suitable bioinks that can accurately replicate the native cellular environment. Decellularized extracellular matrix (dECM) has emerged as a promising candidate due to its inherent biocompatibility, bioactivity, and structural resemblance to native tissues. Decellularization is a crucial process in tissue engineering that involves the removal of cellular components from the extracellular matrix (ECM) to create scaffolds suitable for tissue regeneration. This article provides a review of some decellularization methods, categorizing them into physical, chemical, and biological approaches. The article discusses the advantages and limitations of each method, highlighting the need to balance effective decellularization with the preservation of the ECM’s functional properties. Understanding these methods is critical for developing optimized scaffolds for various tissue engineering applications.

High mechanical performance and multifunctional degraded fucoidan-derived bioink for 3D bioprinting

While 3D bioprinting serves as a powerful tool in the field of tissue engineering, there is still a lack of natural biomaterial inks that simultaneously combine high mechanical performance with multiple biofunctionalities. Here, a single-component natural bioink with high strength and multi-biofunctionality was developed through the simple degradation and methacrylation of natural fucoidan. Hydrothermal degradation significantly decreased the natural fucoidan solution's viscosity by 99.9 %, meeting the necessary viscosity for Digital Light Processing (DLP) 3D printing. Meanwhile, various biofunctionalities of low molecular weight fucoidan obtained through degradation, such as antimicrobial and antioxidant properties, were developed. The resulting bioink exhibited good mechanical performance (compression modulus of 311 kPa), antimicrobial properties (antibacterial rates of 95.5 % and 97.9 % against E. coli and S. aureus, respectively), and antioxidant properties (intracellular ROS inhibition rates of 94.7 %). Using DLP 3D bioprinting, all printed products showed high shape fidelity with exceptional viability and activity of the encapsulated cells. Due to the unique sulfate structure resembling the natural components of chondroitin sulfate, the in vivo tests revealed its efficacy in promoting cartilage defect repair. In conclusion, the novel bioink blending high mechanical performance with multiple biofunctionalities, shows great potential in the 3D printing of tissue and organ regeneration.

Genetic Dissection of ToLCNDV Resistance in Resistant Sources of Cucumis melo.

Tomato leaf curl New Delhi virus (ToLCNDV) is a begomovirus causing significant melon (Cucumis melo) crop losses globally. This study aims to map the ToLCNDV resistance in the PI 414723 melon accession, previously identified and characterized through phenotypic studies, thereby exploring shared genomic regions with the established resistant source WM-7. In the present study, WM-7 and PI 414723 were crossed with the susceptible accessions 'Rochet' and 'Blanco' respectively, to generate F1 hybrids. These hybrids were self-pollinated to generate the populations for mapping the ToLCNDV resistance region and designing markers for marker-assisted selection. Disease evaluation included visual symptom scoring, viral-load quantification and tissue printing. Genotyping-by-sequencing and SNP markers were used for quantitative trait loci (QTL) mapping. For genetic analysis, qPCR and bulked segregant RNA-seq (BSR-seq) were performed. Gene expression was assessed using RNA-seq, and qRT-PCR was used for confirmation. The research narrows the candidate region for resistance in WM-7 and identifies overlapping QTLs on chromosome 11 in PI 414723, found in the region of the DNA primase large subunit. BSR-seq and expression analyses highlight potential regulatory roles of chromosome 2 in conferring resistance. Differential expression was confirmed for six genes in the candidate region on chromosome 2. This study confirms the existence of common resistance genes in PI 414723 and WM-7.

Rapid Customization of Biomimetic Cartilage Scaffold with Stem Cell Capturing and Homing Capabilities for In Situ Inductive Regeneration of Osteochondral Defects

Abstract3D printing of articular cartilage tissue faces challenges like replicating its complex structure, time‐consuming in vitro stem cell culture, and a lack of robust in situ regeneration methods for osteochondral defects (OC). In response, an innovative approach utilizing pre‐designed bioink modular units for one‐step printing and immediate implantation is proposed, circumventing the need for prior in vitro stem cell cultivation. The resulting printed scaffold not only accurately reproduces the three‐layer structure and material gradient of articular cartilage tissue but also attains impressive compressive strength (6.3 MPa) through the reinforcement of hydroxyapatite nanofibers and the establishment of chemical bonds with hydrogels. Moreover, the scaffold integrates stem cell capturing and homing layers on its bottom and top layers via chemical crosslinking aptamer and loading poly (lactic‐co‐glycolic acid) (PLGA) nanospheres encapsulated with stromal cell‐derived factor‐1α (SDF‐1α), respectively. This design enables the specific capture of bone marrow mesenchymal stem cells (BMSCs) in vivo through aptamer interaction, followed by their mobilization to home in on the hyaline cartilage layer via the chemotaxis of SDF‐1α concentration gradient. Within the scaffold's microenvironment, these BMSCs undergo differentiation into distinct cells for each layer, effectively contributing to the repair of OC defects in rabbits.

Masked stereolithography 3D printing of a brain tissue from an MRI data set

3D printing technology holds tremendous promise for applications in the medical field, particularly in the creation of anatomical models. The accuracy of 3D-printed anatomical models is of utmost importance, especially for surgeons engaged in complex surgical planning. The goal of this study was to create an anatomically precise model of brain tissue using data obtained from an MRI scan, with a resin-based Masked Stereolithography 3D printer, and to assess the model's potential uses. The process of creating an anatomical brain model with an SLA 3D printer was divided into six fundamental stages. The 3D printing process for this project necessitated 55.4 g of resin, and the total printing time was 4 h and 56 min. Any liquid resin residues on the brain structure eliminated through cleaning have been conducted using 99.6 % pure isopropyl alcohol. Following the printing phase, the produced model underwent a curing process in a chamber illuminated with ultraviolet light at a wavelength of 405 nm to ensure complete solidification. Once solidification is completed, the dimensions of the brain are measured with a micrometer and compared with those digitally generated from the MRI scan data. The results showed that the overall dimensional error was less than 1 %.

Three-Dimensional Bioprinting: The Ultimate Pinnacle of Tissue Engineering.

Three-dimensional (3D) bioprinting has emerged as a revolutionary additive manufacturing technology that can potentially enable life-changing medical treatments in regenerative medicine. It applies the principles of tissue engineering for the printing of tissues and organs in a layer-by-layer manner. This review focuses on the various 3D bioprinting technologies currently available, the different biomaterials, cells, and growth factors that can be utilized to develop tissue-specific bioinks, the different venues for applying these technologies, and the challenges this technology faces.

Acrylonitrile‐butadiene‐styrene‐based composites for the manufacture of anthropomorphic simulators

AbstractThe development of functional compounds for extrusion applied in the additive manufacturing of anthropomorphic simulators is interesting, as it guarantees the manufacture of a 3D model similar to the patient. These simulators find applications in therapies or laboratory tests involving x‐rays. In order to replicate human conditions in these tests, it is essential to create materials that closely match the properties of human tissue, including the smoothness of soft tissues and the hardness of the bone tissue. This study developed ceramic‐polymeric composites, where the tomography intensity of each mixture was measured experimentally. Combinations of acrylonitrile butadiene styrene (ABS) with zirconium oxide and basic bismuth carbonate allowed imitation of bone tissue. The samples containing zirconium oxide and basic bismuth carbonate presented results that exceeded the minimum limit and reached a value close to 2000 Hounsfield units (HU) with 12% basic bismuth carbonate content. Combinations of ABS with hydroxyapatite and aluminum oxide can imitate soft tissues. The use of a surfactant facilitated the mixing of ceramic filler with polymer. Finally, 3D printing of a physical model was performed using a dual extruder printer, allowing simultaneous printing of bone and soft tissue components.Highlights Material mimicking x‐ray attenuation similar to bone tissue. Relation between 3D printing porosity and intensity in Hounsfield unit. Computed tomography tests on a 3D printed anthropomorphic phantom. Creating a 3D model from a Computed Tomography scan. Double extruder for 3D printing of two tissue simultaneously.

Bioactive Ions-Loaded Bioinks Primed for 3D Printing of Artificial Tissues

Bioactive Ions-Loaded Bioinks Primed for 3D Printing of Artificial Tissues

Multiscale embedded printing of engineered human tissue and organ equivalents

Creating tissue and organ equivalents with intricate architectures and multiscale functional feature sizes is the first step toward the reconstruction of transplantable human tissues and organs. Existing embedded ink writing approaches are limited by achievable feature sizes ranging from hundreds of microns to tens of millimeters, which hinders their ability to accurately duplicate structures found in various human tissues and organs. In this study, a multiscale embedded printing (MSEP) strategy is developed, in which a stimuli-responsive yield-stress fluid is applied to facilitate the printing process. A dynamic layer height control method is developed to print the cornea with a smooth surface on the order of microns, which can effectively overcome the layered morphology in conventional extrusion-based three-dimensional bioprinting methods. Since the support bath is sensitive to temperature change, it can be easily removed after printing by tuning the ambient temperature, which facilitates the fabrication of human eyeballs with optic nerves and aortic heart valves with overhanging leaflets on the order of a few millimeters. The thermosensitivity of the support bath also enables the reconstruction of the full-scale human heart on the order of tens of centimeters by on-demand adding support bath materials during printing. The proposed MSEP demonstrates broader printable functional feature sizes ranging from microns to centimeters, providing a viable and reliable technical solution for tissue and organ printing in the future.

Prediction of ink flow for 3D bioprinting of tubular tissue based on a back propagation neural network

Based on the development of the 3D vascular printer, the forming process of ink from the nozzle to the rotating rod was studied. In this study, to online detect the ink flow from the nozzle during 3D bioprinting of tubular tissue, we established a geometric model according to the region of interest (ROI) of the ink flow picture of 3D printing of tubular tissue, selected description features of the ink contour, and studied how to select mathematical expressions of the features. Principal component analysis (PCA) was used to simplify the image features into 15 features. We used a back propagation (BP) neural network to predict the printing ink flow. The results show that the error between the actual ink flow rate and the flow rate based on the BP neural network is within 5%. The BP neural network can be used to monitor the quality status of the printing target in real time, evaluate the 3D bioprinting quality online, and predict the printing ink flow for the subsequent improvement of the 3D bioprinting accuracy of tubular tissue.

Magnetic hydroxyapatite nanobelt‐stem cell hybrid spheroids for remotely patterning bone tissues

AbstractThe low survival rate and poor differentiation efficiency of stem cells, as well as the insufficient integration of implanted stem cells, limit the regeneration of bone defects. Here, we have developed magnetic ferroferric oxide‐hydroxyapatite‐polydopamine (Fe3O4‐HAp‐PDA) nanobelts to assemble mesenchymal stem cells (MSCs) into a three‐dimensional hybrid spheroid for patterning bone tissue. These nanobelts, which are featured by their high‐aspect ratio and contain Fe3O4 nanospheres with a PDA coating, can be manipulated by a magnetic field and foster enhanced cell‐nanobelt interactions. This strategy has been demonstrated to be effective for both bone marrow mesenchymal stem cells and adipose‐derived mesenchymal stem cells, enabling remote manipulation of stem cell spheroids and efficient spheroid fusion, which in turn accelerates osteogenic differentiation. Consequently, this methodology serves as an efficient and general tool for bone tissue printing and can potentially overcome the low survival rate and poor differentiation efficiency of stem cells, as well as mismatched interface fusion issues.

Development and optimisation of hydroxyapatite-polyethylene glycol diacrylate hydrogel inks for 3D printing of bone tissue engineered scaffolds

In the event of excessive damage to bone tissue, the self-healing process alone is not sufficient to restore bone integrity. Three-dimensional (3D) printing, as an advanced additive manufacturing technology, can create implantable bone scaffolds with accurate geometry and internal architecture, facilitating bone regeneration. This study aims to develop and optimise hydroxyapatite-polyethylene glycol diacrylate (HA-PEGDA) hydrogel inks for extrusion 3D printing of bone tissue scaffolds. Different concentrations of HA were mixed with PEGDA, and further incorporated with pluronic F127 (PF127) as a sacrificial carrier. PF127 provided good distribution of HA nanoparticle within the scaffolds and improved the rheological requirements of HA-PEGDA inks for extrusion 3D printing without significant reduction in the HA content after its removal. Higher printing pressures and printing rates were needed to generate the same strand diameter when using a higher HA content compared to a lower HA content. Scaffolds with excellent shape fidelity up to 75-layers and high resolution (∼200 µm) with uniform strands were fabricated. Increasing the HA content enhanced the compression strength and decreased the swelling degree and degradation rate of 3D printed HA-PEGDA scaffolds. In addition, the incorporation of HA improved the adhesion and proliferation of human bone mesenchymal stem cells (hBMSCs) onto the scaffolds. 3D printed scaffolds with 2 wt% HA promoted osteogenic differentiation of hBMSCs as confirmed by the expression of alkaline phosphatase activity and calcium deposition. Altogether, the developed HA-PEGDA hydrogel ink has promising potential as a scaffold material for bone tissue regeneration, with excellent shape fidelity and the ability to promote osteogenic differentiation of hBMSCs.

Vibrational spectroscopy identifies myocardial chemical modifications in heart failure with preserved ejection fraction

BackgroundVibrational spectroscopy can be a valuable tool to monitor the markers of cardiovascular diseases. In the present work, we explored the vibrational spectroscopy characteristics of the cardiac tissue in an experimental model of heart failure with preserved ejection fraction (HFpEF). The goal was to detect early cardiac chemical modifications associated with the development of HFpEF.MethodsWe used the Fourier-transform infrared (FTIR) and Raman micro-spectroscopic techniques to provide complementary and objective tools for the histological assessment of heart tissues from an animal model of HFpEF. A new sampling technique was adopted (tissue print on a CaF2 disk) to characterize the extracellular matrix.ResultsSeveral spectroscopic markers (lipids, carbohydrates, and glutamate bands) were recognized in the cardiac ventricles due to the comorbidities associated with the pathology, such as obesity and diabetes. Besides, abnormal collagen cross-linking and a decrease in tryptophan content were observed and related to the stiffening of ventricles and to the inflammatory state which is a favourable condition for HFpEF.ConclusionsBy the analyses of tissues and tissue prints, FTIR and Raman techniques were shown to be highly sensitive and selective in detecting changes in the chemistry of the heart in experimental HFpEF and its related comorbidities. Vibrational spectroscopy is a new approach that can identify novel biomarkers for early detection of HFpEF.

A study on cell viability based on thermal inkjet three-dimensional bioprinting

Thermal inkjet three-dimensional (3D) bioprinting (TIJ) is a biological additive manufacturing technology with high cell viability, fast printing speeds, and low costs. It is widely used in biology, chemistry, and pharmaceuticals. In recent years, remarkable results have been achieved in the printing of biological tissues using TIJ. However, few studies have reported on the relationship between TIJ and cell viability. In particular, there have been no reports relating cell viability and the TIJ input energy. In this work, we aim to determine the relationship between the input pulse, printing frequency, and cell viability from the TIJ working principle and find an optimized pulse waveform to improve cell viability. We propose a novel approach to study cell viability. The state of the droplet is observed while controlling the printing pulse and frequency, and then, the corresponding cell viability is determined. The results show that an increase in the pulse increases the shear stress and temperature in the bio-ink, which reduces the viability of the cells. The shear stress and viability of the printed cells show a corresponding piecewise functional relationship. The cell viability is significantly reduced when the ambient temperature is higher than 40 °C. Increasing the printing frequency reduces the rate of printing heat loss, thereby raising the ambient temperature and impairing cell viability. Finally, the optimized input waveform can increase cell viability by up to about 95%.

3D printing of mechanically functional meniscal tissue equivalents using high concentration extracellular matrix inks

Decellularized extracellular matrix (dECM) has emerged as a promising biomaterial in the fields of tissue engineering and regenerative medicine due to its ability to provide specific biochemical and biophysical cues supportive of the regeneration of diverse tissue types. Such biomaterials have also been used to produce tissue-specific inks and bioinks for 3D printing applications. However, a major limitation associated with the use of such dECM materials is their poor mechanical properties, which limits their use in load-bearing applications such as meniscus regeneration. In this study, native porcine menisci were solubilized and decellularized using different methods to produce highly concentrated dECM inks of differing biochemical content and printability. All dECM inks displayed shear thinning and thixotropic properties, with increased viscosity and improved printability observed at higher pH levels, enabling the 3D printing of anatomically defined meniscal implants. With additional crosslinking of the dECM inks following thermal gelation at pH 11, it was possible to fabricate highly elastic meniscal tissue equivalents with compressive mechanical properties similar to the native tissue. These improved mechanical properties at higher pH correlated with the development of a denser network of smaller diameter collagen fibers. These constructs also displayed repeatable loading and unloading curves when subjected to long-term cyclic compression tests. Moreover, the printing of dECM inks at the appropriate pH promoted a preferential alignment of the collagen fibers. Altogether, these findings demonstrate the potential of 3D printing of highly concentrated meniscus dECM inks to produce mechanically functional and biocompatible implants for meniscal tissue regeneration. This approach could be applied to a wide variety of different biological tissues, enabling the 3D printing of tissue mimics with diverse applications from tissue engineering to surgical planning.

Microbial Polysaccharide-Based Formulation with Silica Nanoparticles; A New Hydrogel Nanocomposite for 3D Printing

Natural polysaccharides are highly attractive biopolymers recommended for medical applications due to their low cytotoxicity and hydrophilicity. Polysaccharides and their derivatives are also suitable for additive manufacturing, a process in which various customized geometries of 3D structures/scaffolds can be achieved. Polysaccharide-based hydrogel materials are widely used in 3D hydrogel printing of tissue substitutes. In this context, our goal was to obtain printable hydrogel nanocomposites by adding silica nanoparticles to a microbial polysaccharide's polymer network. Several amounts of silica nanoparticles were added to the biopolymer, and their effects on the morpho-structural characteristics of the resulting nanocomposite hydrogel inks and subsequent 3D printed constructs were studied. FTIR, TGA, and microscopy analysis were used to investigate the resulting crosslinked structures. Assessment of the swelling characteristics and mechanical stability of the nanocomposite materials in a wet state was also conducted. The salecan-based hydrogels displayed excellent biocompatibility and could be employed for biomedical purposes, according to the results of the MTT, LDH, and Live/Dead tests. The innovative, crosslinked, nanocomposite materials are recommended for use in regenerative medicine.

Innovatív eljárások a sebgyógyításban

Even today, the treatment of wounds, especially chronic wounds, is still a major burden on healthcare. In addition to the many pharmaceutical and surgical interventions, the use of skin substitutes gains ground in this field. Skin substitutes can be considered as artificial tissues, they can help the skin to regenerate and they can model the main functions of the skin, but they have the disadvantage of being mechanically fragile and not durable. They do not contain all the cellular elements of the skin, often only the specific extracellular matrix proteins. New innovative applications aim at creating more complex skin substitutes, using artificial skin tissues created by three-dimensional tissue printing, not only for pre-clinical studies but also for clinical therapeutic use.

Evolution of bioprinting and current applications.

Bioprinting is a very useful tool that has a huge application potential in different fields of science and biotechnology. In medicine, advances in bioprinting are focused on the printing of cells and tissues for skin regeneration and the manufacture of viable human organs, such as hearts, kidneys, and bones. This review provides a chronological overview of some of the most relevant developments of bioprinting technique and its current status. A search was carried out in SCOPUS, Web of Science, and PubMed databases, and a total of 31,603 papers were found, of which 122 were finally chosen for analysis. These articles cover the most important advances in this technique at the medical level, its applications, and current possibilities. Finally, the paper ends with conclusions about the use of bioprinting and our expectations of this technique. This paper presents a review on the tremendous progress of bioprinting from 1998 to the present day, with many promising results indicating that our society is getting closer to achieving the total reconstruction of damaged tissues and organs and thus solving many healthcare-related problems, including the shortage of organ and tissue donors.

Development and Validation of a Duplex RT-qPCR for Detection of Peach Latent Mosaic Viroid and Comparison of Different Nucleic-Acid-Extraction Protocols.

Peach latent mosaic viroid (PLMVd) is an important pathogen that causes disease in peaches. Control of this viroid remains problematic because most PLMVd variants are symptomless, and although there are many detection tests in use, the reliability of PCR-based methods is compromised by the complex, branched secondary RNA structure of the viroid and its genetic diversity. In this study, a duplex RT-qPCR method was developed and validated against two previously published single RT-qPCRs, which were potentially able to detect all known PLMVd variants when used in tandem. In addition, in order to simplify the sample preparation, rapid-extraction protocols based on the use of crude sap or tissue printing were compared with commercially available RNA purification kits. The performance of the new procedure was evaluated in a test performance study involving five participant laboratories. The new method, in combination with rapid-sample-preparation approaches, was demonstrated to be feasible and reliable, with the advantage of detecting all different PLMVd isolates/variants assayed in a single reaction, reducing costs for routine diagnosis.