Three-dimensional Bioprinting Technology Research Articles

Hybrid biofabrication of neurosecretory structures as a model for neurosecretion.

The present study aimed to combine extrusion-based three-dimensional (3D) bioprinting and polymer nanofiber electrospinning technology to fabricate tissue-like structures with neurosecretory function in vitro. Using neurosecretory cells as cell resources, sodium alginate/gelatin/fibrinogen as matrix, polylactic acid/gelatin electrospun nanofibers as diaphragm, and neurosecretory cells-loaded 3D hydrogel scaffolds were bioprinted and then covered with electrospun nanofibers layer-by-layer. The morphology was observed by scanning electron microscopy and transmission electron microscopy (TEM), and the mechanical characteristics and cytotoxicity of the hybrid biofabricated scaffold structure were evaluated. The 3D-bioprinted tissue activity, including cell death and proliferation, was verified. Western blotting and ELISA experiments were used to confirm the cell phenotype and secretory function, while animal in vivo transplantation experiments confirmed the histocompatibility, inflammatory reaction, and tissue remodeling ability of the heterozygous tissue structures. Neurosecretory structures with 3D structures were successfully prepared by hybrid biofabrication in vitro. The mechanical strength of the composite biofabricated structures was significantly higher than that of the hydrogel system (P < 0.05). The survival rate of PC12 cells in the 3D-bioprinted model was 92.849 ± 2.995%. Hematoxylin and eosin-stained pathological sections showed that the cells grew in clumps, and there was no significant difference in the expression of MAP2 and tubulin-β between 3D organoids and PC12 cells. The results of ELISA showed that the PC12 cells in 3D structures retained the ability to continuously secrete noradrenaline and met-enkephalin, and the secretory vesicles around and within the cells could be observed by TEM. In in vivo transplantation, PC12 cells gathered and grew in clusters, maintained high activity, neovascularization, and tissue remodeling in 3D structures. The neurosecretory structures were biofabricated by 3D bioprinting and nanofiber electrospinning in vitro, which had high activity and neurosecretory function. In vivo transplantation of neurosecretory structures showed active proliferation of cells and potential for tissue remodeling. Our research provides a new method for biological manufacture of neurosecretory structures in vitro, which maintains neurosecretory function and lays the foundation for the clinical application of neuroendocrine tissues.

Developments and Clinical Applications of Biomimetic Tissue Regeneration using 3D Bioprinting Technique.

Tissue engineers have made great strides in the past decade thanks to the advent of three-dimensional (3D) bioprinting technology, which has allowed them to create highly customized biological structures with precise geometric design ability, allowing us to close the gap between manufactured and natural tissues. In this work, we first survey the state-of-the-art methods, cells, and materials for 3D bioprinting. The modern uses of this method in tissue engineering are then briefly discussed. Following this, the main benefits of 3D bioprinting in tissue engineering are outlined in depth, including the ability to rapidly prototype the individualized structure and the ability to engineer with a highly controllable microenvironment. Finally, we offer some predictions for the future of 3D bioprinting in the field of tissue engineering.

3D Bioprinting for Pancreas Engineering/Manufacturing

Diabetes is the most common chronic disease in the world, and it brings a heavy burden to people's health. Against this background, diabetic research, including islet functionalization has become a hot topic in medical institutions all over the world. Especially with the rapid development of microencapsulation and three-dimensional (3D) bioprinting technologies, organ engineering and manufacturing have become the main trends for disease modeling and drug screening. Especially the advanced 3D models of pancreatic islets have shown better physiological functions than monolayer cultures, suggesting their potential in elucidating the behaviors of cells under different growth environments. This review mainly summarizes the latest progress of islet capsules and 3D printed pancreatic organs and introduces the activities of islet cells in the constructs with different encapsulation technologies and polymeric materials, as well as the vascularization and blood glucose control capabilities of these constructs after implantation. The challenges and perspectives of the pancreatic organ engineering/manufacturing technologies have also been demonstrated.

A Review on Recent Advances in 3D Bioprinting

Three-dimensional (3D) bioprinting technology has emerged as a powerful bio- fabrication platform for tissue engineering because of its ability to engineer living cells and bio-material based 3D objects. Diverse bio-inks based on synthetic and natural biomaterials have also been created and successfully used for tissue regeneration at the same time. Over the past few decades, the fields of tissue engineering and regenerative medicine, which aim to develop functioning tissue-constructs replicating native tissue for the repair and/or replacement of damaged tissues or entire organs, have advanced quickly. Traditional tissue engineering methods, which include scaffolds, growth factors, and cells, had less success fabricating complicated 3D structures and regenerating organs in vivo, which made them logistically and financially unworkable for clinical applications. In this regard, 3D bioprinting, which is an extended application of additive manufacturing is now being explored for tissue engineering and regenerative medicine as it involves the top-down approach of building the Layer-by- layer construction of complicated tissue, thereby producing precise geometries due to controlled nature of matter deposition with the help of anatomically accurate 3D models of the tissue generated by computer graphics. In this article, we seek to present a thorough analysis of the 3D bioprinting techniques, including ink-jet printing, extrusion printing, stereolithography, and laser aided bioprinting methods. With the exact control of structure, dynamics, and biological elements—such as cells and extracellular matrix (ECM)—3D bioprinting has a tremendous deal of promise to build very complex constructions.

Bacterial nanocellulose-reinforced gelatin methacryloyl hydrogel enhances biomechanical property and glycosaminoglycan content of 3D-bioprinted cartilage.

Tissue-engineered ear cartilage scaffold based on three-dimensional (3D) bioprinting technology presents a new strategy for ear reconstruction in individuals with microtia. Natural hydrogel is a promising material due to its excellent biocompatibility and low immunogenicity. However, insufficient mechanical property required for cartilage is one of the major issues pending to be solved. In this study, the gelatin methacryloyl (GelMA) hydrogel reinforced with bacterial nanocellulose (BNC) was developed to enhance the biomechanical properties and printability of the hydrogel. The results revealed that the addition of 0.375% BNC significantly increased the mechanical properties of the hydrogel and promoted cell migration in the BNC-reinforced hydrogel. Constructs bioprinted with chondrocyte-laden BNC/GelMA hydrogel bio-ink formed mature cartilage in nude mice with higher Young's modulus and glycosaminoglycan content. Finally, an auricle equivalent with a precise shape, high mechanics, and abundant cartilage-specific matrix was developed in vivo. In this study, we developed a potentially useful hydrogel for the manufacture of auricular cartilage grafts for microtia patients.

Research advances on the construction of an ideal scar model in vitro based on innovative tissue engineering technology

The scar brings a huge economic burden and creates a serious psychological shadow for patients. Although the current methods for scar treatment tend to be diversified, the treatment method that can truly achieve the goal of "perfect healing" or "scarless healing" after human skin injury is quite scarce. With the wide application of tissue engineering technologies in medicine research, technologies such as three-dimensional bioprinting, organoid culture, and organ chip technologies are constantly emerging. Disease models in vitro based on these innovative technologies showed more advantages than traditional animal disease models. The article introduces the current hotspot technologies in skin tissue engineering such as organoid culture, three-dimensional bioprinting, and organ chip technologies, focuses on summarizing the three key elements to be mastered for constructing an ideal scar model in vitro, and puts forward the future prospect of constructing an ideal scar model in vitro based on our research team's long-term experience in skin tissue repair and regeneration research.

Three-Dimensional Printed Cellulose for Wound Dressing Applications.

Wounds are skin tissue damage due to trauma. Many factors inhibit the wound healing phase (hemostasis, inflammation, proliferation, and alteration), such as oxygenation, contamination/infection, age, effects of injury, sex hormones, stress, diabetes, obesity, drugs, alcoholism, smoking, nutrition, hemostasis, debridement, and closing time. Cellulose is the most abundant biopolymer in nature which is promising as the main matrix of wound dressings because of its good structure and mechanical stability, moisturizes the area around the wound, absorbs excess exudate, can form elastic gels with the characteristics of bio-responsiveness, biocompatibility, low toxicity, biodegradability, and structural similarity with the extracellular matrix (ECM). The addition of active ingredients as a model drug helps accelerate wound healing through antimicrobial and antioxidant mechanisms. Three-dimensional (3D) bioprinting technology can print cellulose as a bioink to produce wound dressings with complex structures mimicking ECM. The 3D printed cellulose-based wound dressings are a promising application in modern wound care. This article reviews the use of 3D printed cellulose as an ideal wound dressing and their properties, including mechanical properties, permeability aspect, absorption ability, ability to retain and provide moisture, biodegradation, antimicrobial property, and biocompatibility. The applications of 3D printed cellulose in the management of chronic wounds, burns, and painful wounds are also discussed.

Current Advances of Three-Dimensional Bioprinting Application in Dentistry: A Scoping Review.

Three-dimensional (3D) bioprinting technology has emerged as an ideal approach to address the challenges in regenerative dentistry by fabricating 3D tissue constructs with customized complex architecture. The dilemma with current dental treatments has led to the exploration of this technology in restoring and maintaining the function of teeth. This scoping review aims to explore 3D bioprinting technology together with the type of biomaterials and cells used for dental applications. Based on PRISMA-ScR guidelines, this systematic search was conducted by using the following databases: Ovid, PubMed, EBSCOhost and Web of Science. The inclusion criteria were (i) cell-laden 3D-bioprinted construct; (ii) intervention to regenerate dental tissue using bioink, which incorporates living cells or in combination with biomaterial; and (iii) 3D bioprinting for dental applications. A total of 31 studies were included in this review. The main 3D bioprinting technique was extrusion-based approach. Novel bioinks in use consist of different types of natural and synthetic polymers, decellularized extracellular matrix and spheroids with encapsulated mesenchymal stem cells, and have shown promising results for periodontal ligament, dentin, dental pulp and bone regeneration application. However, 3D bioprinting in dental applications, regrettably, is not yet close to being a clinical reality. Therefore, further research in fabricating ideal bioinks with implantation into larger animal models in the oral environment is very much needed for clinical translation.

3D skin models along with skin-on-a-chip systems: A critical review

Skin is the largest organ in human body, and it plays an important role in regulating physiological microenvironments and acts as a barrier to protect human body from harmful intrusions. The demand for fully functional skin models (also called skin equivalents, SE) in an in-vivo mimicking culturing microenvironment has been increased dramatically due to the fast development in skin disease treatments and skin care products. Owing to the emerging of the concept and technology of organ-on-chips along with the three-dimensional (3D) bioprinting technology, 3D skin models and their applications have been fast evolving. In this paper, the advances in the development of 3D skin models along with skin-on-a-chip (SOC) are reviewed and commented. One of the findings with this paper is that the SOC together with the 3D bioprinting technology is promising to construct fully functional 3D skin models in the field of pharmaceutical and cosmetic industries.

3D Printing of Skin Equivalents with Hair Follicle Structures and Epidermal-Papillary-Dermal Layers Using Gelatin/Hyaluronic Acid Hydrogels.

Recent advances in three-dimensional (3D) bioprinting technologies have enabled the fabrication of sophisticated live 3D tissue analogs. Despite the existing hydrogel-based bioinks, the development of advanced bioink materials that can accurately reproduce the composition of a native extracellular matrix and mimic the intrinsic properties of laden cells remains challenging. In this study, 3D printed skin equivalents incorporating hair follicle structures and epidermal/papillary dermal layers were fabricated using gelatin methacryloyl/hyaluronic acid methacryloyl (GelMA/HAMA) bioink. The composition of collagen and glycosaminoglycan in native skin was recapitulated by adjusting the combination of GelMA and HAMA. The GelMA/HAMA bioink exhibited excellent viscoelastic and physicochemical properties, 3D printability, cytocompatibility, and functionality to maintain hair-inductive potency while facilitating spontaneous hair pore development. The results indicate that GelMA/HAMA hydrogels are promising candidates as bioinks for the 3D printing of skin equivalents. Furthermore, they may serve as useful models for skin tissue engineering and regeneration.

Functional Trachea Reconstruction Using 3D-Bioprinted Native-Like Tissue Architecture Based on Designable Tissue-Specific Bioinks.

Functional segmental trachea reconstruction remains a remarkable challenge in the clinic. To date, functional trachea regeneration with alternant cartilage‐fibrous tissue‐mimetic structure similar to that of the native trachea relying on the three‐dimensional (3D) bioprinting technology has seen very limited breakthrough. This fact is mostly due to the lack of tissue‐specific bioinks suitable for both cartilage and vascularized fibrous tissue regeneration, as well as the need for firm interfacial integration between stiff and soft tissues. Here, a novel strategy is developed for 3D bioprinting of cartilage‐vascularized fibrous tissue‐integrated trachea (CVFIT), utilizing photocrosslinkable tissue‐specific bioinks. Both cartilage‐ and fibrous tissue‐specific bioinks created by this study provide suitable printability, favorable biocompatibility, and biomimetic microenvironments for chondrogenesis and vascularized fibrogenesis based on the multicomponent synergistic effect through the hybrid photoinitiated polymerization reaction. As such, the tubular analogs are successfully bioprinted and the ring‐to‐ring alternant structure is tightly integrated by the enhancement of interfacial bonding through the amidation reaction. The results from both the trachea regeneration and the in situ trachea reconstruction demonstrate the satisfactory tissue‐specific regeneration along with realization of mechanical and physiological functions. This study thus illustrates the 3D‐bioprinted native tissue‐like trachea as a promising alternative for clinical trachea reconstruction.

Ovary-derived Decellularized Extracellular Matrix-based Bioink for Fabricating 3D Primary Ovarian Cells-laden Structures for Mouse Ovarian Failure Correction.

Fertility preservation is becoming a clinical duty in practice. Three-dimensional (3D) bioprinting technology is potentially realize ovarian morphological repair and reproductive endocrine function rebuild. There is no published work on 3D bioprinting ovary using a decellularized extracellular matrix (dECM)-based bioink, though dECM is the preferred matrix choice for an artificial ovary. The study aimed to explore swine ovarian dECM-based bioink to fabricate 3D primary ovarian cells (POCs)-laden structures for mouse ovarian failure correction. In this study, the ovarian dECM was converted to dECM-based bioink by dECM solution mixed with a seaweed gelatin blend solution of bioink that was characterized using scanning electron microscopy, circular dichroism, rheology, hematoxylin and eosin staining, and immunohistochemistry. The 3D scaffolds were, then, printed with or without POCs by the extrusion 3D bioprinter. The laden POCs viability was detected with the live/dead assay kit. A female castrated mouse model was established, and the mice were treated with five different methods. The results revealed that the 3D scaffold encapsulating POCs group had more positive signals in neoangiogenesis, cell proliferation and survival than the 3D scaffold group, and ensured sex hormone secretion. Meanwhile, the expression of germ cells in the 3D scaffold encapsulating POCs group was more intensely than the non-printed hydrogel encapsulating POCs group. The work shows that the 3D bioprinting ovary employing ovarian dECM-based bioink is a promising approach for ovarian failure correction.

Research advances of three-dimensional bioprinting technology in urinary system tissue engineering

For the damage and loss of tissues and organs caused by urinary system diseases, the current clinical treatment methods have limitations. Tissue engineering provides a therapeutic method that can replace or regenerate damaged tissues and organs through the research of cells, biological scaffolds and biologically related molecules. As an emerging manufacturing technology, three-dimensional (3D) bioprinting technology can accurately control the biological materials carrying cells, which further promotes the development of tissue engineering. This article reviews the research progress and application of 3D bioprinting technology in tissue engineering of kidney, ureter, bladder, and urethra. Finally, the main current challenges and future prospects are discussed.

Bioprinting of a Hepatic Tissue Model Using Human-Induced Pluripotent Stem Cell-derived Hepatocytes for Drug-Induced Hepatotoxicity Evaluation.

Three-dimensional (3D) bioprinting technology is an effective method for exploring the biological functions of hepatocytes by building biomimetic 3D microenvironments. Various hepatic tissue models have been developed for disease modeling, drug screening, and tissue regeneration using 3D bioprinting technology. Human-induced pluripotent stem cells (hiPSCs) are a promising cell source for the generation of functional hepatocytes for bioprinting. In this study, we introduced hiPSC-derived hepatocytes (hiPSC-Heps) as mature hepatocytes for the bioprinting of a 3D hepatic tissue model. The 3D-printed (3DP) model facilitated the formation of hiPSC-Hep spheroids with higher viability and proliferation than the commonly used non-printed sandwich-cultured model. hiPSC-Heps in the 3DP model exhibited higher mRNA expression of liver-specific functions than those in the two-dimensional-cultured model. Moreover, enhanced secretion of liver function-related proteins, including α-1-antitrypsin, albumin, and blood urea nitrogen, was observed in the 3DP model. For the evaluation of acetaminophen-induced hepatotoxicity, the 3DP model exhibited a favorable drug response with upregulation of the drug metabolism-related gene cytochrome P450-1A2 (CYP1A2). Overall, the bioprinted hepatic tissue model showed great biofunctional and drug-responsive performance, which could be potentially applied in in vitro toxicological studies.

Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering.

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global problem that occurs in 1.5%-5% of emergency patients and may lead to significant job losses. Today, tissue engineering-based approaches, consisting of scaffolds, appropriate cell lines, and biosignals, have become more applicable with the development of three-dimensional (3D) bioprinting technologies. The combination of various hydrogel biomaterials with stem cells, exosomes, or bio-signaling molecules is frequently studied to overcome the existing problems in peripheral nerve regeneration. Accordingly, the production of injectable systems, such as hydrogels, or implantable conduit structures formed by various bioprinting methods has gained importance in peripheral neuro-engineering. Under normal conditions, stem cells are the regenerative cells of the body, and their number and functions do not decrease with time to protect their populations; these are not specialized cells but can differentiate upon appropriate stimulation in response to injury. The stem cell system is under the influence of its microenvironment, called the stem cell niche. In peripheral nerve injuries, especially in neurotmesis, this microenvironment cannot be fully rescued even after surgically binding severed nerve endings together. The composite biomaterials and combined cellular therapies approach increases the functionality and applicability of materials in terms of various properties such as biodegradability, biocompatibility, and processability. Accordingly, this study aims to demonstrate the preparation and use of graphene-based biohybrid hydrogel patterning and to examine the differentiation efficiency of stem cells into nerve cells, which can be an effective solution in nerve regeneration.

Digital light processing-based 3D bioprinting of κ-carrageenan hydrogels for engineering cell-loaded tissue scaffolds

The demand to regenerate biological tissues and organs in patients as an alternative to transplants has motivated the tissue engineering field. Digital light processing (DLP)-based three-dimensional (3D) bioprinting technology enables the rapid fabrication of complex 3D cell-laden scaffolds for tissue engineering applications. Herein, we demonstrate the outstanding printability of photocurable methacrylate-κ-carrageenan (MA-κ-CA) using DLP 3D printing. 3D printed hydrogels with varying concentrations (1–5% w/v) of MA-κ-CA were thoroughly characterized for their swelling, degradation, mechanical, and rheological properties, and suitability for bioprinting with living cells. Viscosity and shear thinning behavior of MA-κ-CA faithfully recapitulate the biomechanical properties of soft human tissues. Encapsulated NIH-3T3 cells show high viability and good proliferation over several days. Furthermore, highly complex 3D hydrogel scaffolds of MA-κ-CA were printed to recapitulate the biological complexity of tissues and organs. This work presents a polysaccharide bioink for preparing tissue scaffolds by DLP 3D bioprinting.

Three-dimensional bioprinting sodium alginate/gelatin scaffold combined with neural stem cells and oligodendrocytes markedly promoting nerve regeneration after spinal cord injury.

Accumulating research has indicated that the transplantation of combined stem cells and scaffolds is an effective method for spinal cord injury (SCI). The development of three-dimensional (3D) bioprinting technology can make the 3D scaffolds combined with cells more accurate and effective for SCI treatment. However, unmyelinated newborn nerve fibers have no nerve signaling conduction, hampering recovery of motor function. In this study, we designed and printed a type of sodium alginate/gelatin scaffold loaded with neural stem cells and oligodendrocytes, which were involved in the formation of the myelin sheaths of neural cell axons. In order to observe the effectiveness of this 3D bioprinting scaffold, we transplanted it into the completely transected rat spinal cord, and then immunofluorescence staining, hematoxylin–eosin staining and behavioral assessment were performed. The results showed that this 3D bioprinting scaffold markedly improved the hindlimb motor function and promoted nerve regeneration. These findings suggested that this novel 3D bioprinting scaffold was a good carrier for cells transplantation, thereby enhancing spinal cord repair following injury.

Three-Dimensional Skin Tissue Printing with Human Skin Cell Lines and Mouse Skin-Derived Epidermal and Dermal Cells.

Since the skin covers most surfaces of the body, it is susceptible to damage, which can be fatal depending on the degree of injury to the skin because it defends against external attack and protects internal structures. Various types of artificial skin are being studied for transplantation to repair damaged skin, and recently, the production of replaceable skin using three-dimensional (3D) bioprinting technology has also been investigated. In this study, skin tissue was produced using a 3D bioprinter with human skin cell lines and cells extracted from mouse skin, and the printing conditions were optimized. Gelatin was used as a bioink, and fibrinogen and alginate were used for tissue hardening after printing. Printed skin tissue maintained a survival rate of 90% or more when cultured for 14 days. Culture conditions were established using 8 mM calcium chloride treatment and the skin tissue was exposed to air to optimize epidermal cell differentiation. The skin tissue was cultured for 14 days after differentiation induction by this optimized culture method, and immunofluorescent staining was performed using epidermal cell differentiation markers to investigate whether the epidermal cells had differentiated. After differentiation, loricrin, which is normally found in terminally differentiated epidermal cells, was observed in the cells at the tip of the epidermal layer, and cytokeratin 14 was expressed in the lower cells of the epidermis layer. Collectively, this study may provide optimized conditions for bioprinting and keratinization for three-dimensional skin production.

Neural stem cell-laden 3D bioprinting of polyphenol-doped electroconductive hydrogel scaffolds for enhanced neuronal differentiation

The development of three-dimensional (3D) bioprinting technology opens a door for constructing bionic nerve tissue scaffolds on demand for nerve injury repair. However, the electrical insulation of the current nerve tissue scaffolds fabricated by 3D bioprinting hinders the bioelectrical signal transmission between cells, limiting the therapeutic effect in nerve tissue repair. To address this issue, we have developed a neural stem cell (NSC)-laden 3D bioprinted electroconductive hydrogel (ECH) scaffold, composed of modified poly(3,4-ethylenedioxythiophene) (PEDOT), gelatin methacrylate/polyethylene glycol diacrylate hydrogel matrix, and NSCs, to promote the electron propagation in the scaffold for enhanced nerve regeneration. In our strategy, PEDOT is modified by doping with chondroitin sulfate and tannic acid (TA) to improve its water-solubility and electric property. With moderate mechanical strength and good electroconductivity, the 3D ECH scaffold provides a benign conductive microenvironment for the adhesion, growth, and proliferation of the encapsulated NSCs. Clearly, in the 3D ECH scaffolds, the NSCs not only maintain high cell viability (>90%) after bioprinting, but also tend to differentiate into neurons with extended neurites. This study testifies for the first time the effect of polyphenol structure belonging to TA on neuronal differentiation of NSCs, and offers a new insight into designing electroconductive biomaterials to induce neuronal regeneration for nerve injury repair and neurodegenerative disease therapy.

A focused review on three-dimensional bioprinting technology for artificial organ fabrication.

Three-dimensional (3D) bioprinting technology has attracted a great deal of interest because it can be easily adapted to many industries and research sectors, such as biomedical, manufacturing, education, and engineering. Specifically, 3D bioprinting has provided significant advances in the medical industry, since such technology has led to significant breakthroughs in the synthesis of biomaterials, cells, and accompanying elements to produce composite living tissues. 3D bioprinting technology could lead to the immense capability of replacing damaged or injured tissues or organs with newly dispensed cell biomaterials and functional tissues. Several types of bioprinting technology and different bio-inks can be used to replicate cells and generate supporting units as complex 3D living tissues. Bioprinting techniques have undergone great advancements in the field of regenerative medicine to provide 3D printed models for numerous artificial organs and transplantable tissues. This review paper aims to provide an overview of 3D-bioprinting technologies by elucidating the current advancements, recent progress, opportunities, and applications in this field. It highlights the most recent advancements in 3D-bioprinting technology, particularly in the area of artificial organ development and cancer research. Additionally, the paper speculates on the future progress in 3D-bioprinting as a versatile foundation for several biomedical applications.