3D Bioprinting Method Research Articles

3D bioprinting for auricular reconstruction: A review and future perspectives

Congenital abnormalities or acquired trauma to the auricle can result in a need for ear reconstruction and negatively impact a person’s quality of life. Autografting, alloplastic implants, and prostheses are available to treat these issues, but each requires multiple surgical stages and has limitations and complications. Three-dimensional (3D) bioprinting promises to allow the creation of living, patient-specific ear substitutes that could reduce operative morbidity. In this review, we evaluate the current state of 3D bioprinting methods through a systematic search and review of 27 studies, aiming to examine this emerging technology within the context of existing reconstructive options. The included studies were all non-randomized experimental studies, except for a single pilot clinical trial. Most of these studies involved both in vitro and in vivo experiments demonstrating the potential of 3D bioprinting to create functional and anatomically accurate engineered cartilaginous frameworks for surgical implantation. Various ways of optimizing printing were identified, from choosing the most suitable material and cell type for the construct to addressing scaffold deformation and shrinkage issues. 3D printing has the potential to revolutionize reconstructive ear surgery by creating functional and aesthetically pleasing auricles. While more research into printing parameters, bioinks, cell types, and materials could optimize results, the next step is to conduct long-term in vivo clinical trials in humans.

Bioprinted hASC-laden cell constructs with mechanically stable and cell alignment cue for tenogenic differentiation.

3D bioprinting is a technology that enables the precise and controlled deposition of cells and an artificial extracellular matrix (ECM) to create functional tissue constructs. However, current 3D bioprinting methods still struggle to obtain mechanically stable and unique cell-morphological structures, such as fully aligned cells. In this study, we propose a new 3D bioprinting approach that utilizes a high concentration of bioink without cells to support mechanical properties and drag flow to fully align cells in a thin bath filled with cell-laden bioink, resulting in a hybrid cell-laden construct with a mechanical stable and fully aligned cell structure. To demonstrate the feasibility of this approach, we used it to fabricate a cell-laden construct using human adipose stem cells (hASCs) for tendon tissue engineering. To achieve appropriate processing conditions, various factors such as the bioink concentration, nozzle moving speed, and volume flow rate were considered. To enhance the biocompatibility of the cell-laden construct, we used porcine decellularized tendon ECM.In vitrocellular responses, including tenogenic differentiation of the fabricated hybrid cell structures with aligned or randomly distributed cells, were evaluated using hASCs. In addition, the mechanical properties of the hybrid cell-laden construct could be adjusted by controlling the concentration of the mechanically reinforcing strut using methacrylated tendon-decellularized extracellular matrix. Based on these results, the hybrid cell-laden structure has the potential to be a highly effective platform for the alignment of musculoskeletal tissues.

Current Progress of 3D Bioprinting of Cardiac Tissues

Heart disease is the leading cause of death worldwide. Heart transplants are used when treating end-stage heart disease. The main problem faced by heart transplantation is the scarcity of donors. Therefore, finding a way to replace the donor heart remains a major medical challenge. In recent years, 3D bioprinting has often been used in tissue engineering, and it has achieved results in the preparation of many types of human tissues. Therefore, it is regarded as a promising method for alleviating donor heart scarcity. In this article, we summarize the current research progress of 3D bioprinting of cardiac tissue, and after introducing several 3D bioprinting methods, bioinks, and 3D bioprinting steps for heart tissue, we proposed our perspectives on 3D bioprinting, discussed several current challenges, and analysed the future of development.

Modeling and analysis of post-processing conditions on 4D-bioprinting of deformable hydrogel-based biomaterial inks

Deformable structures have been actively developed for several biomedical applications including drug delivery and tissue engineering using 3D-bioprinting methods. However, structural shape-transformation usually consists of a series of bending behaviors in response to external stimuli, which require complex aggregation and multi-materials design. To overcome these complexities, this work explores an alternative approach using only a single hydrogel-based material to realize such bending mechanisms. Numerical simulations are first implemented to realize bending deformation by spatially assigning distinct material parameters to different sections of the structure. The bending phenomenon is also shown experimentally using hydrogel-based biomaterial inks. Specifically, a deformable structure is fabricated by finely controlling different post-processing conditions such as cooling time, crosslinking duration, and heating rate during swelling to mimic the effect of different material parameters. Moreover, the bending deformation can be further analyzed using computer vision methods to inversely determine the desired material coefficients in the simulation. Relationships among bending mechanisms, material parameters, and post-processing procedures are found and shown to affect the final bending orientation. These results yield insightful approaches to the inverse design of functional biomedical devices with desired deformation behavior.

3D Printing Approaches to Engineer Cardiac Tissue.

Bioengineering of functional cardiac tissue composed of primary cardiomyocytes has great potential for myocardial regeneration and in vitro tissue modeling. 3D bioprinting was developed to create cardiac tissue in hydrogels that can mimic the structural, physiological, and functional features of native myocardium. Through a detailed review of the 3D printing technologies and bioink materials used in the creation of a heart tissue, this article discusses the potential of engineered heart tissues in biomedical applications. In this review, we discussed the recent progress in 3D bioprinting strategies for cardiac tissue engineering, including bioink and 3D bioprinting methods as well as examples of engineered cardiac tissue such as in vitro cardiac models and vascular channels. 3D printing is a powerful tool for creating in vitro cardiac tissues that are structurally and functionally similar to real tissues. The use of human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) enables the generation of patient-specific tissues. These tissues have the potential to be used for regenerative therapies, disease modeling, and drug testing.

Advancements in Research on Constructing Physiological and Pathological Liver Models and Their Applications Utilizing Bioprinting Technology.

In recent decades, significant progress has been made in liver tissue engineering through the use of 3D bioprinting technology. This technology offers the ability to create personalized biological structures with precise geometric design capabilities. The complex and multifaceted nature of liver diseases underscores the need for advanced technologies to accurately mimic the physiological and mechanical characteristics, as well as organ-level functions, of liver tissue in vitro. Bioprinting stands out as a superior option over traditional two-dimensional cell culture models and animal models due to its stronger biomimetic advantages. Through the use of bioprinting, it is possible to create liver tissue with a level of structural and functional complexity that more closely resembles the real organ, allowing for more accurate disease modeling and drug testing. As a result, it is a promising tool for restoring and replacing damaged tissue and organs in the field of liver tissue engineering and drug research. This article aims to present a comprehensive overview of the progress made in liver tissue engineering using bioprinting technology to provide valuable insights for researchers. The paper provides a detailed account of the history of liver tissue engineering, highlights the current 3D bioprinting methods and bioinks that are widely used, and accentuates the importance of existing in vitro liver tissue models based on 3D bioprinting and their biomedical applications. Additionally, the article explores the challenges faced by 3D bioprinting and predicts future trends in the field. The progress of 3D bioprinting technology is poised to bring new approaches to printing liver tissue in vitro, while offering powerful tools for drug development, testing, liver disease modeling, transplantation, and regeneration, which hold great academic and practical significance.

3D Bioprinting in Otolaryngology: A Review.

The evolution of tissue engineering and 3D bioprinting has allowed for increased opportunities to generate musculoskeletal tissue grafts that can enhance functional and aesthetic outcomes in otolaryngology-head and neck surgery. Despite literature reporting successes in the fabrication of cartilage and bone scaffolds for applications in the head and neck, the full potential of this technology has yet to be realized. Otolaryngology as a field has always been at the forefront of new advancements and technology and is well poised to spearhead clinical application of these engineered tissues. In this review, current 3D bioprinting methods are described and an overview of potential cell types, bioinks, and bioactive factors available for musculoskeletal engineering using this technology is presented. The otologic, nasal, tracheal, and craniofacial bone applications of 3D bioprinting with a focus on engineered graft implantation in animal models to highlight the status of functional outcomes in vivo; a necessary step to future clinical translation are reviewed. Continued multidisciplinary efforts between material chemistry, biological sciences, and otolaryngologists will play a key role in the translation of engineered, 3D bioprinted constructs for head and neck surgery.

3D-Printed disposable nozzles for cost-efficient extrusion-based 3D bioprinting

3D bioprinting has significantly impacted tissue engineering with its capability to create intricate structures with complex geometries that were difficult to replicate through traditional manufacturing techniques. Extrusion-based 3D bioprinting methods tend to be limited when creating complex structures using bioinks of low viscosity. However, the capacity for creating multi-material structures that have distinct properties could be unlocked through the mixture of two solutions before extrusion. This could be used to generate architectures with varying levels of stiffness and hydrophobicity, which could be utilized for regenerative medicine applications. Moreover, it allows for combining proteins and other biological materials in a single 3D-bioprinted structure. This paper presents a standardized fabrication method of disposable nozzle connectors (DNC) for 3D bioprinting with hydrogel-based materials. This method entails 3D printing connectors with dual inlets and a single outlet to mix the material internally. The connectors are compatible with conventional Luer lock needles, offering an efficient solution for nozzle replacement. IVZK (Ac-Ile-Val-Cha-Lys-NH2) peptide-based hydrogel materials were used as a bioink with the 3D-printed DNCs. Extrusion-based 3D bioprinting was employed to print shapes of varying complexities, demonstrating potential in achieving high print resolution, shape fidelity, and biocompatibility. Post-printing of human neonatal dermal fibroblasts, cell viability, proliferation, and metabolic activity were observed, which demonstrated the effectiveness of the proposed design and process for 3D bioprinting using low-viscosity bioinks.

Cartilage 3D bioprinting for rhinoplasty using adipose-derived stem cells as seed cells: Review and recent advances.

Nasal deformities due to various causes affect the aesthetics and use of the nose, in which case rhinoplasty is necessary. However, the lack of cartilage for grafting has been a major problem and tissue engineering seems to be a promising solution. 3D bioprinting has become one of the most advanced tissue engineering methods. To construct ideal cartilage, bio-ink, seed cells, growth factors and other methods to promote chondrogenesis should be considered and weighed carefully. With continuous progress in the field, bio-ink choices are becoming increasingly abundant, from a single hydrogel to a combination of hydrogels with various characteristics, and more 3D bioprinting methods are also emerging. Adipose-derived stem cells (ADSCs) have become one of the most popular seed cells in cartilage 3D bioprinting, owing to their abundance, excellent proliferative potential, minimal morbidity during harvest and lack of ethical considerations limitations. In addition, the co-culture of ADSCs and chondrocytes is commonly used to achieve better chondrogenesis. To promote chondrogenic differentiation of ADSCs and construct ideal highly bionic tissue-engineered cartilage, researchers have used a variety of methods, including adding appropriate growth factors, applying biomechanical stimuli and reducing oxygen tension. According to the process and sequence of cartilage 3D bioprinting, this review summarizes and discusses the selection of hydrogel and seed cells (centered on ADSCs), the design of printing, and methods for inducing the chondrogenesis of ADSCs.

Application and Progress of Cultured Models of Gallbladder Carcinoma.

Gallbladder carcinoma (GBC) is a malignant tumor of the biliary system that is aggressive, difficult to detect early, and has a low surgical resection rate and poor prognosis. Appropriate in vitro growth models are expected to focus on the study of the biological behavior and assess treatment effects. Nonetheless, cancer initiation, progression, and invasion include spatiotemporal changes and changes in the cell microenvironment intracellular communication, and intracellular molecules, making the development of in vitro growth models very challenging. Recent advances in biomaterial methods and tissue engineering, particularly advances in bioprinting procedures, have paved the way for advances in the creative phase of in vitro cancer research. To date, an increasing number of cultured models of gallbladder disease have emerged, such as two-dimensional (2D) GBC growth cell cultures, three-dimensional (3D) GBC growth cell cultures, xenograft models, and 3D bioprinting methods. These models can serve as stronger platforms, focusing on tumor growth initiation, the association with the microenvironment, angiogenesis, motility, aggression, and infiltration. Bioprinted growth models can also be used for high-throughput drug screening and validation, as well as translational opportunities for individual cancer therapy. This study focused on the exploration, progress, and significance of the development of GBC cultural models. We present our views on the shortcomings of existing models, investigate new innovations, and plan future improvements and application possibilities for cancer models.

Four-Dimensional Printing of Stimuli-Responsive Hydrogel-Based Soft Robots.

The present protocol describes the creation of four-dimensional (4D), time-dependent, shape-changeable, stimuli-responsive soft robots using a three-dimensional (3D) bio-printing method. Recently, 4D printing techniques have been extensively proposed as innovative new methods for developing shape-transformable soft robots. In particular, 4D time-dependent shape transformation is an essential factor in soft robotics because it allows effective functions to occur at the right time and place when triggered by external cues, such as heat, pH, and light. In line with this perspective, stimuli-responsive materials, including hydrogels, polymers, and hybrids, can be printed to realize smart shape-transformable soft robotic systems. The current protocol can be used to fabricate thermally responsive soft grippers composed of N-isopropylacrylamide (NIPAM)-based hydrogels, with overall sizes ranging from millimeters to centimeters in length. It is expected that this study will provide new directions for realizing intelligent soft robotic systems for various applications in smart manipulators (e.g., grippers, actuators, and pick-and-place machines), healthcare systems (e.g., drug capsules, biopsy tools, and microsurgeries), and electronics (e.g., wearable sensors and fluidics).

Bioprinting Methods for Fabricating InVitro Tubular Blood Vessel Models.

Dysfunctional blood vessels are implicated in various diseases, including cardiovascular diseases, neurodegenerative diseases, and cancer. Several studies have attempted to prevent and treat vascular diseases and understand interactions between these diseases and blood vessels across different organs and tissues. Initial studies were conducted using 2-dimensional (2D) invitro and animal models. However, these models have difficulties in mimicking the 3D microenvironment in human, simulating kinetics related to cell activities, and replicating human pathophysiology; in addition, 3D models involve remarkably high costs. Thus, invitro bioengineered models (BMs) have recently gained attention. BMs created through biofabrication based on tissue engineering and regenerative medicine are breakthrough models that can overcome limitations of 2D and animal models. They can also simulate the natural microenvironment in a patient- and target-specific manner. In this review, we will introduce 3D bioprinting methods for fabricating bioengineered blood vessel models, which can serve as the basis for treating and preventing various vascular diseases. Additionally, we will describe possible advancements from tubular to vascular models. Last, we will discuss specific applications, limitations, and future perspectives of fabricated BMs.

3D bioprinting tumor models mimic the tumor microenvironment for drug screening.

Cancer is a severe threat to human life and health and represents the main cause of death globally. Drug therapy is one of the primary means of treating cancer; however, most anticancer medications do not proceed beyond preclinical testing because the conditions of actual human tumors are not effectively mimicked by traditional tumor models. Hence, bionic in vitro tumor models must be developed to screen for anticancer drugs. Three-dimensional (3D) bioprinting technology can produce structures with built-in spatial and chemical complexity and models with accurately controlled structures, a homogeneous size and morphology, less variation across batches, and a more realistic tumor microenvironment (TME). This technology can also rapidly produce such models for high-throughput anticancer medication testing. This review describes 3D bioprinting methods, the use of bioinks in tumor models, and in vitro tumor model design strategies for building complex tumor microenvironment features using biological 3D printing technology. Moreover, the application of 3D bioprinting in vitro tumor models in drug screening is also discussed.

Advances and Innovations of 3D Bioprinting Skin.

Three-dimensional (3D) bioprinted skin equivalents are highlighted as the new gold standard for alternative models to animal testing, as well as full-thickness wound healing. In this review, we focus on the advances and innovations of 3D bioprinting skin for skin regeneration, within the last five years. After a brief introduction to skin anatomy, 3D bioprinting methods and the remarkable features of recent studies are classified as advances in materials, structures, and functions. We will discuss several ways to improve the clinical potential of 3D bioprinted skin, with state-of-the-art printing technology and novel biomaterials. After the breakthrough in the bottleneck of the current studies, highly developed skin can be fabricated, comprising stratified epidermis, dermis, and hypodermis with blood vessels, nerves, muscles, and skin appendages. We hope that this review will be priming water for future research and clinical applications, that will guide us to break new ground for the next generation of skin regeneration.

3D-Printed Pectin/Carboxymethyl Cellulose/ZnO Bio-Inks: Comparative Analysis with the Solution Casting Method.

Bio-inks consisting of pectin (Pec), carboxymethyl cellulose (CMC), and ZnO nanoparticles (ZnO) were used to prepare films by solution casting and 3D-printing methods. Field emission scanning electron microscopy (FE-SEM) was conducted to observe that the surface of samples made by 3D bioprinter was denser and more compact than the solution cast samples. In addition, Pec/CMC/ZnO made by 3D-bioprinter (Pec/CMC/ZnO-3D) revealed enhanced water vapor barrier, hydrophobicity, and mechanical properties. Pec/CMC/ZnO-3D also showed strong antimicrobial activity within 12 h against S. aureus and E. coli O157: H7 bacterial strains compared to the solution cast films. Further, the nanocomposite bio-inks used for 3D printing did not show cytotoxicity towards normal human dermal fibroblast (NDFB) cells but enhanced the fibroblast proliferation with increasing exposure concentration of the sample. The study provided two important inferences. Firstly, the 3D bioprinting method can be an alternative, better, and more practical method for fabricating biopolymer film instead of solution casting, which is the main finding of this work defining its novelty. Secondly, the Pec/CMC/ZnO can potentially be used as 3D bio-inks to fabricate functional films or scaffolds and biomedical applications.

3D Bioprinting: Introduction and Recent Advancement

In the additive manufacturing method known as 3D bioprinting, living cells and nutrients are joined with organic and biological components to produce synthetic structures that resemble natural human tissues. To put it another way, bioprinting is a type of 3D printing that can create anything from bone tissue and blood vessels to living tissues for a range of medical purposes, including tissue engineering and drug testing and discovery. During the bioprinting process, a solution of a biomaterial or a mixture of several biomaterials in the hydrogel form, usually encapsulating the desired cell types, which are termed as bioink, is used for creating tissue constructs. This bioink can be cross-linked or stabilised during or immediately after bioprinting to generate the designed construct's final shape, structure, and architecture. This report thus offers a comprehensive review of the 3D bioprinting technology along with associated 3D bioprinting methods including ink-jet printing, extrusion printing, stereolithography, laser-assisted bioprinting and microfluidic techniques. We also focus on the types of materials, cell source, maturing, the implant of various representative tissue and organs, including blood vessels, bone and cartilage as well as recent advancements related to 3D bioprinting technology.

Three-dimensional bioprinting of mucoadhesive scaffolds for the treatment of oral mucosal lesions; an in vitro study

BackgroundChronic oral lesions could be a part of some diseases, including mucocutaneous diseases, immunobullous diseases, gastrointestinal diseases, and graft versus host diseases. Systemic steroids are an effective treatment, but they cause unfavorable and even severe systemic side effects. Discontinuation of systemic corticosteroids or other immunosuppressive drugs leads to relapse, confirming the importance of long-term corticosteroid use. The present study aims to fabricate a mucoadhesive scaffold using three-dimensional (3D) bioprinting for sustained drug delivery in oral mucosal lesions to address the clinical need for alternative treatment, especially for those who do not respond to routine therapy.Methods3D bioprinting method was used for the fabrication of the scaffolds. Scaffolds were fabricated in three layers; adhesive/drug-containing, backing, and middle layers. For evaluation of the release profile of the drug, artificial saliva was used as the release medium. Mucoadhesive scaffolds were analyzed using a scanning electron microscope (SEM) and SEM surface reconstruction. The pH of mucoadhesive scaffolds and swelling efficacy were measured using a pH meter and Enslin dipositive, respectively. A microprocessor force gauge was used for the measurement of tensile strength. For the evaluation of the cytotoxicity, oral keratinocyte cells' survival rate was evaluated by the MTT method. Folding endurance tests were performed using a stable microsystem texture analyzer and analytic probe mini tensile grips.ResultsAll scaffolds had the same drug release trend; An initial rapid explosive release during the first 12 h, followed by a gradual release. The scaffolds showed sustained drug release and continued until the fourth day. The pH of the surface of the scaffolds was 5.3–6.3, and the rate of swelling after 5 h was 28 ± 3.2%. The tensile strength of the scaffolds containing the drug was 7.8 ± 0.12 MPa. The scaffolds were non-irritant to the mucosa, and the folding endurance of the scaffolds was over three hundred times.ConclusionThe scaffold fabricated using the 3D bioprinting method could be suitable for treating oral mucosal lesions.

Leveraging ultra-low interfacial tension and liquid-liquid phase separation in embedded 3D bioprinting.

Many recently developed 3D bioprinting strategies operate by extruding aqueous biopolymer solutions directly into a variety of different support materials constituted from swollen, solvated, aqueous, polymer assemblies. In developing these 3D printing methods and materials, great care is often taken to tune the rheological behaviors of both inks and 3D support media. By contrast, much less attention has been given to the physics of the interfaces created when structuring one polymer phase into another in embedded 3D printing applications. For example, it is currently unclear whether a dynamic interfacial tension between miscible phases stabilizes embedded 3D bioprinted structures as they are shaped while in a liquid state. Interest in the physics of interfaces between complex fluids has grown dramatically since the discovery of liquid-liquid phase separation (LLPS) in living cells. We believe that many new insights coming from this burst of investigation into LLPS within biological contexts can be leveraged to develop new materials and methods for improved 3D bioprinting that leverage LLPS in mixtures of biopolymers, biocompatible synthetic polymers, and proteins. Thus, in this review article, we highlight work at the interface between recent LLPS research and embedded 3D bioprinting methods and materials, and we introduce a 3D bioprinting method that leverages LLPS to stabilize printed biopolymer inks embedded in a bioprinting support material.

Three-Dimensional In Vitro Cell Culture Models for Efficient Drug Discovery: Progress So Far and Future Prospects.

Despite tremendous advancements in technologies and resources, drug discovery still remains a tedious and expensive process. Though most cells are cultured using 2D monolayer cultures, due to lack of specificity, biochemical incompatibility, and cell-to-cell/matrix communications, they often lag behind in the race of modern drug discovery. There exists compelling evidence that 3D cell culture models are quite promising and advantageous in mimicking in vivo conditions. It is anticipated that these 3D cell culture methods will bridge the translation of data from 2D cell culture to animal models. Although 3D technologies have been adopted widely these days, they still have certain challenges associated with them, such as the maintenance of a micro-tissue environment similar to in vivo models and a lack of reproducibility. However, newer 3D cell culture models are able to bypass these issues to a maximum extent. This review summarizes the basic principles of 3D cell culture approaches and emphasizes different 3D techniques such as hydrogels, spheroids, microfluidic devices, organoids, and 3D bioprinting methods. Besides the progress made so far in 3D cell culture systems, the article emphasizes the various challenges associated with these models and their potential role in drug repositioning, including perspectives from the COVID-19 pandemic.

Development and Application of Three-Dimensional Bioprinting Scaffold in the Repair of Spinal Cord Injury.

Spinal cord injury (SCI) is a disabling and destructive central nervous system injury that has not yet been successfully treated at this stage. Three-dimensional (3D) bioprinting has become a promising method to produce more biologically complex microstructures, which fabricate living neural constructs with anatomically accurate complex geometries and spatial distributions of neural stem cells, and this is critical in the treatment of SCI. With the development of 3D printing technology and the deepening of research, neural tissue engineering research using different printing methods, bio-inks, and cells to repair SCI has achieved certain results. Although satisfactory results have not yet been achieved, they have provided novel ideas for the clinical treatment of SCI. Considering the potential impact of 3D bioprinting technology on neural studies, this review focuses on 3D bioprinting methods widely used in SCI neural tissue engineering, and the latest technological applications of bioprinting of nerve tissues for the repair of SCI are discussed. In addition to introducing the recent progress, this work also describes the existing limitations and highlights emerging possibilities and future prospects in this field.