Bioprinting Research Articles

3D Bioprinting a Novel Skin Co-Culture Model Using Human Keratinocytes and Fibroblasts.

3D bioprinting can generate the organized structures found in human skin for a variety of biological, medical, and pharmaceutical applications. Challenges in bioprinting skin include printing different types of cells in the same construct while maintaining their viability, which depends on the type of bioprinter and bioinks used. This study evaluated a novel 3D bioprinted skin model containing human keratinocytes (HEKa) and human dermal fibroblasts (HDF) in co-culture (CC) using a high-viscosity fibrin-based bioink produced using the BioX extrusion-based bioprinter. The constructs containing HEKa or HDF cells alone (control groups) and in CC were evaluated at 1, 10, and 20 days after bioprinting for viability, immunocytochemistry for specific markers (K5 and K10 for keratinocytes; vimentin and fibroblast specific protein [FSP] for fibroblasts). The storage, loss modulus, and viscosity properties of the constructs were also assessed to compare the effects of keratinocytes and fibroblasts individually and combined, providing important insights when bioprinting skin. Our findings revealed significantly higher cell viability in the CC group compared to individual keratinocyte and fibroblast groups, suggesting the combined cell presence enhanced survival rates. Additionally, proliferation rates of both cell types remained consistent over time, indicating non-competitive growth within the construct. Interestingly, keratinocytes exhibited a greater impact on the viscoelastic properties of the construct compared to fibroblasts, likely due to their larger size and arrangement. These insights contribute to optimizing bioprinting strategies for skin tissue engineering and emphasize the important role of different cell types in 3D skin models.

A natural composite hydrogel laden with mesenchymal stromal cells for osteochondral repair: Comparison between casting and 3D bioprinting

A natural composite hydrogel laden with mesenchymal stromal cells for osteochondral repair: Comparison between casting and 3D bioprinting

3D Bioprinting Strategies for Melatonin‐Loaded Polymers in Bone Tissue Engineering

AbstractBone pathologies are still among the most challenging issues for orthopedics. Over the past decade, different methods are developed for bone repair. In addition to advanced surgical and graft techniques, polymer‐based biomaterials, bioactive glass, chitosan, hydrogels, nanoparticles, and cell‐derived exosomes are used for bone healing strategies. Owing to their variation and promising advantages, most of these methods are not translated into clinical practice. Three dimensonal (3D) bioprinting is an additive manufacturing technique that has become a next‐generation biomaterial technique adapted for anatomic modeling, artificial tissue or organs, grafting, and bridging tissues. Polymer‐based biomaterials are mostly used for the controlled release of various drugs, therapeutic agents, mesenchymal stem cells, ions, and growth factors. Polymers are now among the most preferable materials for 3D bioprinting. Melatonin is a well‐known antioxidant with many osteoinductive properties and is one of the key hormones in the brain–bone axis. 3D bioprinted melatonin‐loaded polymers with unique lipophilic, anti‐inflammatory, antioxidant, and osteoinductive properties for filling large bone gaps following fractures or congenital bone deformities may be developed in the future. This study summarized the benefits of 3D bioprinted and polymeric materials integrated with melatonin for sustained release in bone regeneration approaches.

Hydrogel composition and mechanical stiffness of 3D bioprinted cell-loaded scaffolds promote cartilage regeneration

ObjectiveTo investigate the impact of different component ratios and mechanical stiffness of the gelatin-sodium alginate composite hydrogel scaffold, fabricated through 3D bioprinting, on the viability and functionality of chondrocytes.MethodsThree different concentrations of hydrogel, designated as low, medium, and high, were prepared. The rheological properties of the hydrogel were characterized to optimize printing parameters. Subsequently, the printability and shape fidelity of the cell-loaded hydrogel scaffolds were statistically evaluated, and the chondrocyte viability was observed. Dynamic mechanical analysis was conducted to measure the modulus, thereby assessing the scaffold’s stiffness. Following a 21-day culture period, RT-PCR, histological staining, and immunostaining were employed to assess chondrocyte activity, chondrosphere aggregates formation, and cartilage matrix production.ResultsBased on rheological analysis, optimal printing temperatures for each group were determined as 27.8°C, 28.5°C, and 30°C. The optimized printing parameters could ensure the molding effect of the scaffolds on the day of printing, with the actual grid area of the scaffolds was close to the theoretical grid area. And the scaffolds exhibited good cell viability (93.24% ± 0.99%, 92.04% ± 1.49%, and 88.46% ± 1.53%). After 7 days of culture, the medium and high concentration groups showed no significant change in grid area compared to the day of printing (p > 0.05), indicating good morphological fidelity. As the hydrogel’s bicomponent ratio increased, both the storage modulus and loss modulus increased, while the loss factor remained relatively constant. The highest number of chondrocytes-formed chondrosphere aggregates in the medium concentration group was observed by light microscopy. RT-PCR results indicated significantly higher expression levels of chondrogenic genes SOX9, Agg, and Col-II in the low and medium concentration groups compared to the high concentration group (p < 0.05). Histological staining results showed that the middle concentration group formed the highest number of typical cartilage lacunae.ConclusionThe aforementioned results indicate that in 3D bioprinted cell-loaded GA-SA composite hydrogel scaffolds, the scaffolds with the composition ratio (10:3) and mechanical stiffness (∼155 kPa) exhibit sustained morphological fidelity, effectively preserve the hyaline phenotype of chondrocytes, and are more conducive to cartilage regeneration.

Incorporating silica nanoparticles with silver patches into alginate-based bioinks for 3D bioprinting

Abstract Alginate dialdehyde-gelatin (ADA-GEL) hydrogels are being studied in bioprinting for their combination of cell adhesion and printability. To incorporate specific functionalities and to improve printability, different additives to ADA-GEL inks are being proposed. Here, a novel type of functional nanoparticles comprising silver patches on silica cores was incorporated into ADA-GEL bioinks. Silver patchy particles (SPPs) were present both on the surface and interior of printed structures. Incorporation of SPPs improved printability of ADA-GEL inks and supported osteosarcoma (MG-63) cells over 7 days of culture. SPPs represent a valuable additive for ADA-GEL hydrogels, being attractive to develop bioinks with advanced functionalities. Graphical abstract

Noncontact 3D Bioprinting of Proteinaceous Microarrays for Highly Sensitive Immunofluorescence Detection within Clinical Samples.

Immunofluorescence assays are extensively used for the detection of disease-associated biomarkers within patient samples for direct diagnosis. Unfortunately, these 2D microarrays suffer from low repeatability and fail to attain the low limits of detection (LODs) required to accurately discern disease progression for clinical monitoring. While three-dimensional microarrays with increased biorecognition molecule density stand to circumvent these limitations, their viscous component materials are not compatible with current microarray fabrication protocols. Herein, we introduce a platform for 3D microarray bioprinting, wherein a two-step printing approach enables the high-throughput fabrication of immunosorbent hydrogels. The hydrogels are composed entirely of cross-linked proteins decorated with clinically relevant capture antibodies. Compared to two-dimensional microarrays, these proteinaceous microarrays offer 3-fold increases in signal intensity. When tested with clinically relevant biomarkers, ultrasensitive single-plex and multiplex detection of interleukin-6 (LOD 0.3 pg/mL) and tumor necrosis factor receptor 1 (LOD 1 pg/mL) is observed. When challenged with clinical samples, these hydrogel microarrays consistently discern elevated levels of interleukin-6 in blood plasma derived from patients with systemic blood infections. Given their easy-to-implement, high-throughput fabrication, and ultrasensitive detection, these three-dimensional microarrays will enable better clinical monitoring of disease progression, yielding improved patient outcomes.

Remote-Controlled Gene Delivery in Coaxial 3D-Bioprinted Constructs using Ultrasound-Responsive Bioinks

Abstract Introduction Coaxial 3D bioprinting has advanced the formation of tissue constructs that recapitulate key architectures and biophysical parameters for in-vitro disease modeling and tissue-engineered therapies. Controlling gene expression within these structures is critical for modulating cell signaling and probing cell behavior. However, current transfection strategies are limited in spatiotemporal control because dense 3D scaffolds hinder diffusion of traditional vectors. To address this, we developed a coaxial extrusion 3D bioprinting technique using ultrasound-responsive gene delivery bioinks. These bioink materials incorporate echogenic microbubble gene delivery particles that upon ultrasound exposure can sonoporate cells within the construct, facilitating controllable transfection. Methods Phospholipid-coated gas-core microbubbles were electrostatically coupled to reporter transgene plasmid payloads and incorporated into cell-laden alginate bioinks at varying particle concentrations. These bioinks were loaded into the coaxial nozzle core for extrusion bioprinting with CaCl2 crosslinker in the outer sheath. Resulting bioprints were exposed to 2.25 MHz focused ultrasound and evaluated for microbubble activation and subsequent DNA delivery and transgene expression. Results Coaxial printing parameters were established that preserved the stability of ultrasound-responsive gene delivery particles for at least 48 h in bioprinted alginate filaments while maintaining high cell viability. Successful sonoporation of embedded cells resulted in DNA delivery and robust ultrasound-controlled transgene expression. The number of transfected cells was modulated by varying the number of focused ultrasound pulses applied. The size region over which DNA was delivered was modulated by varying the concentration of microbubbles in the printed filaments. Conclusions Our results present a successful coaxial 3D bioprinting technique designed to facilitate ultrasound-controlled gene delivery. This platform enables remote, spatiotemporally-defined genetic manipulation in coaxially bioprinted tissue constructs with important applications for disease modeling and regenerative medicine.

3D bioprinted in vitro epilepsy models for pharmacological evaluation in temporal lobe epilepsy.

This study introduces a novel in vitro model for intractable temporal lobe epilepsy (TLE) utilizing 3D bioprinting technology, aiming to replicate the complex neurobiological characteristics of TLE more accurately . Primary neural cell constructs were fabricated and subjected to epileptiform-inducing conditions, fostering synaptic proliferation and neuronal loss. Systematically electrophysiological and immunofluorescent analyses indicated that significant synaptic connectivity and sustained epileptiform activities within the constructs akin to those observed in human epilepsy models. Notably, the model responded to treatments with phenytoin and tetrodotoxin, illustrating its potential utility in drug response kinetics studies. Furthermore, we performed drug permeability simulations using COMSOL Multiphysics to analyze the diffusion characteristics of these drugs within the constructs. These results confirm that our 3D bioprinted neural model provides a physiologically relevant and ethically sustainable platform, which is beneficial for studying TLE mechanisms and developing therapeutic strategies with high accuracy and clinical relevance.&#xD.

3D Bioprinting in Cancer Modeling and Biomedicine: From Print Categories to Biological Applications

3D Bioprinting in Cancer Modeling and Biomedicine: From Print Categories to Biological Applications

Double-Network Hydrogel 3D BioPrinting Biocompatible with Fibroblast Cells for Tissue Engineering Applications

The present study examines the formulation of a biocompatible hydrogel bioink for 3D bioprinting, integrating poly(ethylene glycol) diacrylate (PEGDA) and sodium alginate (SA) using a double-network approach. These materials were chosen for their synergistic qualities, with PEGDA contributing to mechanical integrity and SA ensuring biocompatibility. Fibroblast cells were included in the bioink and printed with a Reg4Life bioprinter employing micro-extrusion technology. The optimisation of printing parameters included needle size and flow velocities. This led to precise structure development and yielded results with a negligible deviation in printed angles and better control of line widths. The rheological characteristics of the bioink were evaluated, demonstrating appropriate viscosity and shear-thinning behaviour for efficient extrusion. The mechanical characterisation revealed an average compressive modulus of 0.38 MPa, suitable for tissue engineering applications. The printability of the bioink was further confirmed through the evaluations of morphology and diffusion rates, confirming structural integrity. Biocompatibility assessments demonstrated a high cell viability rate of 82.65% following 48 h of incubation, supporting the bioink’s suitability for facilitating cell survival. This study introduced a reliable technique for producing tissue-engineered scaffolds that exhibit outstanding mechanical characteristics and cell viability, highlighting the promise of PEGDA–SA hydrogels in bioprinting applications.

Development of printable bacterial nanocellulose bioinks for bioprinting applications

This research has developed a printable bioink formulated with bacterial nanocellulose (BNC) and sodium alginate. The bioink composition offers a unique combination of printability, mechanical strength, and high BNC content. A low-cost 3D bioprinting platform, with in-situ crosslinking capabilities, was designed and fabricated to facilitate the creation of complex constructs. Rheological analysis revealed shear-thinning behaviour, a critical property for efficient 3D bioprinting. The printed constructs exhibited exceptional resolution, printability, and self-supporting features at BNC loadings up to 72 wt.% in dried structures. The optimal ink composition was found, containing 7.8 wt.% BNC and 3 wt.% sodium alginate. These findings highlight the potential for this BNC-alginate bioink for bio-fabrication applications demanding both printability and high biomaterial content.Graphical

Dynamically cultured, differentiated bovine adipose-derived stem cell spheroids as building blocks for biofabricating cultured fat

Cultured or cultivated meat, animal muscle, and fat tissue grown in vitro, could transform the global meat market, reducing animal suffering while using fewer resources than traditional meat production and no antimicrobials at all. To ensure the appeal of cultured meat to future customers, cultured fat is essential for achieving desired mouthfeel, taste, and texture, especially in beef. In this work we show the establishment of primary bovine adipose-derived stem cell spheroids in static and dynamic suspension culture. Spheroids are successfully differentiated using a single-step protocol. Differentiated spheroids from dynamic cultures maintain stability and viability during 3D bioprinting in edible gellan gum. Also, the fatty acid composition of differentiated spheroids is significantly different from control spheroids. The cells are cultured antibiotic-free to minimize the use of harmful substances. This work presents a stable and bioprintable building block for cultured fat with a high cell density in a 3D dynamic cell culture system.

Dynamically Crosslinked Hydrogels Composed of Carboxymethyl Chitosan and Tannic Acid for Sustained Release of Encapsulated Protein

AbstractPolysaccharide‐based hydrogels are promising for biomedical applications such as drug delivery owing to their biocompatibility, biodegradability, and bioactivities. In particular, there is a need for hydrogels with injectability for minimally invasive therapies and controlled sustained release for sustained drug effect. Hydrogels made from carboxymethyl chitosan (CMC) and tannic acid (TA) (CMC‐TA) contain dynamic covalent bonds owing to autoxidation between CMC and TA, and interact with proteins via TA, elucidation of the detailed mechanism of dynamic covalent bonding in CMC‐TA hydrogels will facilitate stable loading and zero‐order release of proteins. Herein, the physical properties of oxidized CMC‐TA (Oxi‐CMC‐TA) prepared for the encapsulation and sustained release of proteins are explored. Following incubation at 37 °C for 24 h, CMC‐TA undergoes secondary crosslinking by autoxidation to produce Oxi‐CMC‐TA. Notably, CMC‐TA is viscoelastic and shear‐thinning, allowing for injection and 3D bioprinting. Indeed, CMC‐TA can be 3D‐printed with green fluorescent protein (GFP) encapsulated in its matrix. Oxi‐CMC‐TA also exhibits an affinity for protein owing to its gallol groups, enabling Oxi‐CMC‐TA to collect GFP in aqueous solutions against a concentration gradient. Moreover, Oxi‐CMC‐TA releases GFP over 15 days. Injectable, 3D‐printable, protein‐collecting, and zero‐order sustained‐releasing Oxi‐CMC‐TA has the potential to make a significant contribution to drug delivery.

Assembly of a tumor microtissue by stacking normal and cancer spheroids on Kenzan using a bio-3D printer to monitor dynamic cancer cell invasion in the microtissue

In the present study, a tumor microtissue was assembled by precisely stacking normal and cancer cell spheroids on Kenzan (microneedle array) using a spheroid stacking type bio-3D printer. This is the first study to non-invasively observe the dynamic behavior of GFP-tagged cancer cell invasion in the microtissue assembled by a spheroid stacking type bio-3D printer. First, the cancer cell spheroid was prepared using 10 % cancer cells (MCF-7 expressing GFP) and 90 % normal cells (70 % HNDF and 20 % HUVEC). The normal cell spheroid was prepared using 100 % normal cells (80 % HNDF and 20 % HUVEC). The tumor microtissue was then assembled on Kenzan by placing 1 cancer cell spheroid in the center position of the microtissue and 8 normal cell spheroids around it. 9 spheroids stacked on Kenzan were fused into 1 tumor microtissue after 24 hours of culture. The green fluorescence derived from cancer cells spread from the central position to the entire area of the tumor microtissue. The spread dynamics of cancer cell-derived GFP fluorescence can be used as a simple measure to evaluate cancer cell migration/invasion and response to anticancer drugs.

3D Nanofiber-Assisted Embedded Extrusion Bioprinting for Oriented Cardiac Tissue Fabrication.

Three-dimensional (3D) bioprinting technology stands out as a promising tissue manufacturing process to control the geometry precisely with cell-loaded bioinks. However, the isotropic culture environment within the bioink and the lack of topographical cues impede the formation of oriented cardiac tissue. To overcome this limitation, we present a novel method named 3D nanofiber-assisted embedded bioprinting (3D-NFEP) to fabricate cardiac tissue with an oriented morphology. Aligned 3D nanofiber scaffolds were fabricated by divergence electrospinning, which provided structural support for printing of the low-viscosity bioink and structural induction to cardiomyocytes. Cells adhered to the aligned fibers after hydrogel degradation, and a high degree of cell alignment was observed. This technology was also demonstrated as a feasible solution for multilayer cell printing. Therefore, 3D-NFEP was demonstrated as a promising method for bioprinting oriented cardiac tissue with low-viscosity bioink and is expected to be applied for structured and cardiac tissue engineering.

3D-BIOPRINTING OF BONE TISSUE IN VITRO И IN SITU

Advanced techniques in bone tissue regeneration include innovative achievements in the field of tissue engineering, among which 3D bioprinting technology holds a special place. This method demonstrates significant progress, driven by its numerous advantages, confirmed through extensive scientific research. 3D bioprinting offers the capability of precise printing of bone transplants with specified geometry, incorporating necessary biomaterials such as cells and growth factors, which facilitates effective osteoinduction, osteoconduction, and vascularization. This review article analyzes various modern methods of bone tissue restoration, including the use of autotransplants, allotransplants, and synthetic bone transplants. Particular attention is paid to the latest innovations in 3D bioprinting, opening prospects for creating individualized bone tissues characterized by a high degree of biocompatibility and minimizing the need for invasive procedures. Additionally, the article presents the concept of in situ bioprinting, developed by Weiss and his colleagues in 2007, as a method of direct printing of bio-inks directly in the area of damage in a living organism. This progressive approach provides high precision in the restoration of tissue defects, regardless of their complexity and geometric features. Over the last decade, several successful clinical cases of using printed structures have been registered, demonstrating the significance of 3D bioprinting in treating life-threatening diseases, as well as in accelerating the regeneration process and creating tissue transplants for subsequent implantation.

Molecules in Motion: Unravelling the Dynamics of Vascularization Control in Tissue Engineering.

Significant progress has been made in tissue engineering (TE), aiming at providing personalized solutions and overcoming the current limitations of traditional tissue and organ transplantation. 3D bioprinting has emerged as a transformative technology in the field, able to mimic key properties of the natural architecture of the native tissues. However, most successes in the area are still limited to avascular or thin tissues due to the difficulties in controlling the vascularization of the engineered tissues. To address this issue, several molecules, biomaterials, and cells with pro- and anti-angiogenic potential have been intensively investigated. Furthermore, different bioreactors capable to provide a dynamic environment for in vitro vascularization control have been also explored. The present review summarizes the main molecules and TE strategies used to promote and inhibit vascularization in TE, as well as the techniques used to deliver them. Additionally, it also discusses the current challenges in 3D bioprinting and in tissue maturation to control in vitro/in vivo vascularization. Currently, this field of investigation is of utmost importance and may open doors for the design and development of more precise and controlled vascularization strategies in TE.

Comparison study on hyaline cartilage versus fibrocartilage formation in a pig model by using 3D-bioprinted hydrogel and hybrid constructs.

Cartilage tissue engineering (CTE) with the help of engineered constructs has shown promise for the regeneration of hyaline cartilage, where fibrocartilage may also be formed due to the biomechanical loading resulting from the host weight and movement. Previous studies have primarily reported on hyaline cartilage formation in vitro and/or in small animals, while leaving the fibrocartilage formation undiscovered. In this paper, we, at the first time, present a comparison study on hyaline cartilage versus fibrocartilage formation in a large animal model of pig by using two constructs (namely hydrogel and hybrid ones) engineered by means of three-dimensional (3D) bioprinting. Both hydrogel and hybrid constructs were printed from the bioink of alginate (2.5%) and ATDC5 cells (chondrogenic cells at a cell density of 5x106 cells/mL), with the difference in that in the hybrid construct, there was a polycaprolactone (PCL) strand printed between every two bioink strands, which were strategically designed to shield the force imposed on the cells within the bioink strands. Both hydrogel and hybrid constructs were implanted into the chondral defects created in the articular cartilage of weight-bearing portions of pig stifle joints; the cartilage formation was examined at one- and three-months post-implantation, respectively, by means of Safranin O, Trichrome, immunofluorescent staining, and synchrotron radiation-based (SR) inline phase contrast imaging microcomputed tomography (inline-PCI-CT). Glycosaminoglycan (GAG) and collagen type 2 (Col2) secretion were used to evaluate the hyaline cartilage formation, while collagen type 1 (Col1) was used to indicate fibrocartilage given that Col1 is low in hyaline cartilage but high in fibrocartilage. Our results revealed that cartilage formation was enhanced over time in both hydrogel and hybrid constructs; particularly, the hydrogel construct exhibited more cartilage formation at both one- and three-months post-implantation, while hybrid constructs tended to have less fibrocartilage formed in a long time period. Also, the result from the inline-PCI-CT revealed that the inline-PCI-CT was able to provide not only the information seen in other histology images, but also high-resolution details of biomaterials and regenerating cartilage. This would represent a significant advance toward the non-invasive assessment of cartilage formation regeneration within large animal models and eventually in human patients.

Enhanced Myogenic Differentiation of Human Adipose‐Derived Stem Cells via Integration of 3D Bioprinting and In Situ Shear‐Based Blade Coating

AbstractConventional treatments for volumetric muscle loss (VML) encounter limitations, such as donor site constraints and exploration of tissue engineering methods. Here, the fabrication of human adipose stem cell (hASC)‐laden cell constructs are proposed using a 3D bioprinting technique supported by blade casting. This process induces mechanotransduction to activate stem cell activities, including proliferation and myogenic differentiation. The printing conditions are optimized by assessing the effects of various process parameters on mechanotransduction signaling pathways. Notably, blade‐assisted bioprinting under carefully selected parameters enhanced the in vitro myogenic activity of the fabricated hASC constructs. Moreover, in vivo evaluation in mice with VML defects demonstrate that shear‐induced bioconstructs effectively restored lost functionalities and muscle volume compared to those of normally bioprinted cell constructs. The results show the potential of integrating bioprinting with hASC‐based therapies to enhance muscle regeneration and functional recovery, offering a meaningful platform for future tissue engineering approaches for VML treatment.

3D Bioprinting for Engineered Tissue Constructs and Patient-Specific Models: Current Progress and Prospects in Clinical Applications.

Advancements in bioprinting technology are driving the creation of complex, functional tissue constructs for use in tissue engineering and regenerative medicine. Various methods, including extrusion, jetting, and light-based bioprinting, have their unique advantages and drawbacks. Over the years, researchers and industry leaders have made significant progress in enhancing bioprinting techniques and materials, resulting in the production of increasingly sophisticated tissue constructs. Despite this progress, challenges still need to be addressed in achieving clinically relevant, human-scale tissue constructs, presenting a hurdle to widespread clinical translation. However, with ongoing interdisciplinary research and collaboration, the field is rapidly evolving and holds promise for personalized medical interventions. Continued development and refinement of bioprinting technologies have the potential to address complex medical needs, enabling the development of functional, transplantable tissues and organs, as well as advanced in vitro tissue models.