Bioprinting Techniques Research Articles

Biomimetic scaffold and 3D bioprinting in dental application: A review.

Biomimetic scaffold and 3D bioprinting technologies have emerged as promising avenues in tissue engineering and regenerative medicine, offering innovative approaches to address the limitations of conventional tissue engineering methods. This review article provides a comprehensive overview of recent advancements, challenges, and future prospects in the field of biomimetic scaffold fabrication and 3D bioprinting techniques.

Advancements in 3D bioprinting for precision medicine: Enhancing patient-derived organoids and extracellular vesicle applications in inflammatory diseases

The integration of three-dimensional (3D) bioprinting with cultures of patient-derived organoids represents a transformative advancement in precision medicine, enabling the creation of anatomically accurate and physiologically relevant tissue models. These models are pivotal for personalized disease modeling, therapeutic development, and regenerative medicine. 3D bioprinting enhances the structural and functional fidelity of organoids, thereby improving their application in drug screening and disease intervention strategies. Additionally, this technology facilitates the study of extracellular vesicles (EVs) released from patient-derived organoids (PDOs), which are rich in bioactive molecules and play crucial roles in modulating immune responses. Particularly in chronic inflammatory conditions, these organoid-derived EVs are instrumental in cellular communication and improve our understanding of disease mechanisms beyond traditional biopsies. This review focuses on the latest developments in 3D bioprinting techniques for organoids and EVs, highlighting their potential to revolutionize the treatment of inflammatory diseases through precision medicine.

Focus on seed cells: stem cells in 3D bioprinting of corneal grafts.

Corneal opacity is one of the leading causes of severe vision impairment. Corneal transplantation is the dominant therapy for irreversible corneal blindness. However, there is a worldwide shortage of donor grafts and consequently an urgent demand for alternatives. Three-dimensional (3D) bioprinting is an innovative additive manufacturing technology for high-resolution distribution of bioink to construct human tissues. The technology has shown great promise in the field of bone, cartilage and skin tissue construction. 3D bioprinting allows precise structural construction and functional cell printing, which makes it possible to print personalized full-thickness or lamellar corneal layers. Seed cells play an important role in producing corneal biological functions. And stem cells are potential seed cells for corneal tissue construction. In this review, the basic anatomy and physiology of the natural human cornea and the grafts for keratoplasties are introduced. Then, the applications of 3D bioprinting techniques and bioinks for corneal tissue construction and their interaction with seed cells are reviewed, and both the application and promising future of stem cells in corneal tissue engineering is discussed. Finally, the development trends requirements and challenges of using stem cells as seed cells in corneal graft construction are summarized, and future development directions are suggested.

Advanced biomedical and electronic dual-function skin patch created through microfluidic-regulated 3D bioprinting

Artificial skin involves multidisciplinary efforts, including materials science, biology, medicine, and tissue engineering. Recent studies have aimed at creating skins that are multifunctional, intelligent, and capable of regenerating tissue. In this work, we present a specialized 3D printing ink composed of polyurethane and bioactive glass (PU-BG) and prepare dual-function skin patch by microfluidic-regulated 3D bioprinting (MRBP) technique. The MRBP endows the skin patch with a highly controlled microstructure and superior strength. Besides, an asymmetric tri-layer is further constructed, which promotes cell attachment and growth through a dual transport mechanism based on hydrogen bonds and gradient structure from hydrophilic to superhydrophilic. More importantly, by combining the features of biomedical skin with electronic skin (e-skin), we achieved a biomedical and electronic dual-function skin patch. In vivo experiments have shown that this skin patch can enhance hemostasis, resist bacterial growth, stimulate the regeneration of blood vessels, and accelerate the healing process. Meanwhile, it also mimics the sensory functions of natural skin to realize signal detection, where the sensitivity reached up to 5.87 kPa−1, as well as cyclic stability (over 500 cycles), a wide detection range of 0–150 kPa, high pressure resolution of 0.1 % under the pressure of 100 kPa. This work offers a versatile and effective method for creating dual-function skin patches and provide new insights into wound healing and tissue repair, which have significant implications for clinical applications.

Droplet 3D cryobioprinting for fabrication of free‐standing and volumetric structures

AbstractDroplet‐based bioprinting has shown remarkable potential in tissue engineering and regenerative medicine. However, it requires bioinks with low viscosities, which makes it challenging to create complex 3D structures and spatially pattern them with different materials. This study introduces a novel approach to bioprinting sophisticated volumetric objects by merging droplet‐based bioprinting and cryobioprinting techniques. By leveraging the benefits of cryopreservation, we fabricated, for the first time, intricate, self‐supporting cell‐free or cell‐laden structures with single or multiple materials in a simple droplet‐based bioprinting process that is facilitated by depositing the droplets onto a cryoplate followed by crosslinking during revival. The feasibility of this approach is demonstrated by bioprinting several cell types, with cell viability increasing to 80%–90% after up to 2 or 3 weeks of culture. Furthermore, the applicational capabilities of this approach are showcased by bioprinting an endothelialized breast cancer model. The results indicate that merging droplet and cryogenic bioprinting complements current droplet‐based bioprinting techniques and opens new avenues for the fabrication of volumetric objects with enhanced complexity and functionality, presenting exciting potential for biomedical applications.

3D-bioprinted bone for regenerative medicine: Current concepts and future perspectives

The shortages in human tissue and organ donors have made clinical therapy relatively challenging. Therefore, research has been initiated over the last decades to develop artificial tissues and organs, particularly from cell and tissue cultures. Three-dimensional (3D) bioprinting is a recent technology capable of building structures for implantation, and these constructs closely resemble native tissues, such as skin, liver, connective tissues, and supportive tissues (bone and cartilage). In this review, we briefly introduce the structure, function, and development of bone tissues, followed by a detailed discussion on 3D bioprinting techniques, materials, and their recent advancements for clinical applications.

Breathing new life into tissue engineering: exploring cutting-edge vascularization strategies for skin substitutes.

Tissue-engineered skin substitutes (TESS) emerged as anew therapeutic option to improve skin transplantation. However, establishing an adequate and rapid vascularization in TESS is a critical factor for their clinical application and successful engraftment in patients. Therefore, several methods have been applied to improve the vascularization of skin substitutes including (i) modifying the structural and physicochemical properties of dermal scaffolds; (ii) activating biological scaffolds with growth factor-releasing systems or gene vectors; and (iii) developing prevascularized skin substitutes by loading scaffolds with capillary-forming cells. This review provides a detailed overview of the most recent and important developments in the vascularization strategies for skin substitutes. Onthe one hand, we present cell-based approaches using stem cells, microvascular fragments, adipose tissue derived stromal vascular fraction, endothelial cells derived from blood and skin as well as other pro-angiogenic stimulation methods. On the other hand, we discuss how distinct 3D bioprinting techniques and microfluidics, miRNA manipulation, cell sheet engineering and photosynthetic scaffolds like GelMA, can enhance skin vascularization for clinical applications. Finally, we summarize and discuss the challenges and prospects of the currently available vascularization techniques that may serve as a steppingstone to a mainstream application of skin tissue engineering.

Advancement in Cancer Vasculogenesis Modeling through 3D Bioprinting Technology.

Cancer vasculogenesis is a pivotal focus of cancer research and treatment given its critical role in tumor development, metastasis, and the formation of vasculogenic microenvironments. Traditional approaches to investigating cancer vasculogenesis face significant challenges in accurately modeling intricate microenvironments. Recent advancements in three-dimensional (3D) bioprinting technology present promising solutions to these challenges. This review provides an overview of cancer vasculogenesis and underscores the importance of precise modeling. It juxtaposes traditional techniques with 3D bioprinting technologies, elucidating the advantages of the latter in developing cancer vasculogenesis models. Furthermore, it explores applications in pathological investigations, preclinical medication screening for personalized treatment and cancer diagnostics, and envisages future prospects for 3D bioprinted cancer vasculogenesis models. Despite notable advancements, current 3D bioprinting techniques for cancer vasculogenesis modeling have several limitations. Nonetheless, by overcoming these challenges and with technological advances, 3D bioprinting exhibits immense potential for revolutionizing the understanding of cancer vasculogenesis and augmenting treatment modalities.

3D Bioprinting of Biomimetic Alginate/Gelatin/Chondroitin Sulfate Hydrogel Nanocomposites for Intrinsically Chondrogenic Differentiation of Human Mesenchymal Stem Cells.

3D-printed hydrogel scaffolds biomimicking the extracellular matrix (ECM) are key in cartilage tissue engineering as they can enhance the chondrogenic differentiation of mesenchymal stem cells (MSCs) through the presence of active nanoparticles such as graphene oxide (GO). Here, biomimetic hydrogels were developed by cross-linking alginate, gelatin, and chondroitin sulfate biopolymers in the presence of GO as a bioactive filler, with excellent processability for developing bioactive 3D printed scaffolds and for the bioprinting process. A novel bioink based on our hydrogel with embedded human MSCs presented a cell survival rate near 100% after the 3D bioprinting process. The effects of processing and filler concentration on cell differentiation were further quantitatively evaluated. The nanocomposited hydrogels render high MSC proliferation and viability, exhibiting intrinsic chondroinductive capacity without any exogenous factor when used to print scaffolds or bioprint constructs. The bioactivity depended on the GO concentration, with the best performance at 0.1 mg mL-1. These results were explained by the rational combination of the three biopolymers, with GO nanoparticles having carboxylate and sulfate groups in their structures, therefore, biomimicking the highly negatively charged ECM of cartilage. The bioactivity of this biomaterial and its good processability for 3D printing scaffolds and 3D bioprinting techniques open up a new approach to developing novel biomimetic materials for cartilage repair.

Bioprinting in cardiovascular medicine: Possibilities, challenges, and future perspectives for low and middle-income countries.

Cardiovascular diseases (CVD) stemming from various factors significantly impact the quality of life (QoL) and are prevalent with high mortality rates in both developed and developing countries. In cases where pharmacotherapy proves insufficient and end-stage disease ensues, a heart transplant/surgical repair becomes the only feasible treatment option. However, challenges such as a limited supply of heart donors, complications associated with rejection, and issues related to medication compliance introduce an additional burden to healthcare services and adversely affect patient outcomes. The emergence of bioprinting has facilitated advancements in creating structures, including ventricles, valves, and blood vessels. Notably, the development of myocardial/cardiac patches through bioprinting has offered a promising avenue for revascularizing, strengthening, and regenerating ventricles. Employment loss in developing countries as a circumstance of disability or death can severely impact a family's well-being and means for sustainable living. Innovations by means of life sustaining treatment options can provide hope for the impoverished and help reduce disability burden on the economy of low- to middle-income countries (LMICs). Such developments can have a significant impact that can last for generations, especially in developing countries. In this review, the authors delve into various types of bioprinting techniques, exploring their possibilities, challenges, and potential future applications in treating various end-stage cardiovascular conditions in LMICs.

Anisotropic hydrogel scaffold by flow-induced stereolithography 3D printing technique

Essential organs, such as the heart and liver, contain a unique porous network that allows oxygen and nutrients to be exchanged, with distinct random to ordered regions displaying varying degrees of strength. A novel technique, referred to here as flow-induced lithography, was developed. This technique generates tunable anisotropic three-dimensional (3D) structures. The ink for this bioprinting technique was made of titanium dioxide nanorods (Ti) and kaolinite nanoclay (KLT) dispersed in a GelMA/PEGDA polymeric suspension. By controlling the flow rate, aligned particle microstructures were achieved in the suspensions. The application of UV light to trigger the polymerization of the photoactive prepolymer freezes the oriented particles in the polymer network. Because the viability test was successful in shearing suspensions containing cells, the flow-induced lithography technique can be used with both acellular scaffolds and cell-laden structures. Fabricated hydrogels show outstanding mechanical properties resembling human tissues, as well as significant cell viability (> 95 %) over one week. As a result of this technique and the introduction of bio-ink, a novel approach has been pioneered for developing anisotropic tissue implants utilizing low-viscosity biomaterials.

Advancing Engineered Plant Living Materials through Tobacco BY-2 Cell Growth and Transfection within Tailored Granular Hydrogel Scaffolds.

In this study, an innovative approach is presented in the field of engineered plant living materials (EPLMs), leveraging a sophisticated interplay between synthetic biology and engineering. We detail a 3D bioprinting technique for the precise spatial patterning and genetic transformation of the tobacco BY-2 cell line within custom-engineered granular hydrogel scaffolds. Our methodology involves the integration of biocompatible hydrogel microparticles (HMPs) primed for 3D bioprinting with Agrobacterium tumefaciens capable of plant cell transfection, serving as the backbone for the simultaneous growth and transformation of tobacco BY-2 cells. This system facilitates the concurrent growth and genetic modification of tobacco BY-2 cells within our specially designed scaffolds. These scaffolds enable the cells to develop into predefined patterns while remaining conducive to the uptake of exogenous DNA. We showcase the versatility of this technology by fabricating EPLMs with unique structural and functional properties, exemplified by EPLMs exhibiting distinct pigmentation patterns. These patterns are achieved through the integration of the betalain biosynthetic pathway into tobacco BY-2 cells. Overall, our study represents a groundbreaking shift in the convergence of materials science and plant synthetic biology, offering promising avenues for the evolution of sustainable, adaptive, and responsive living material systems.

Integrating single-cell and spatial analysis reveals MUC1-mediated cellular crosstalk in mucinous colorectal adenocarcinoma.

Mucinous colorectal adenocarcinoma (MCA) is a distinct subtype of colorectal cancer (CRC) with the most aggressive pattern, but effective treatment of MCA remains a challenge due to its vague pathological characteristics. An in-depth understanding of transcriptional dynamics at the cellular level is critical for developing specialised MCA treatment strategies. We integrated single-cell RNA sequencing and spatial transcriptomics data to systematically profile the MCA tumor microenvironment (TME), particularly the interactome of stromal and immune cells. In addition, a three-dimensional bioprinting technique, canonical ex vivo co-culture system, and immunofluorescence staining were further applied to validate the cellular communication networks within the TME. This study identified the crucial intercellular interactions that engaged in MCA pathogenesis. We found the increased infiltration of FGF7+/THBS1+ myofibroblasts in MCA tissues with decreased expression of genes associated with leukocyte-mediated immunity and T cell activation, suggesting a crucial role of these cells in regulating the immunosuppressive TME. In addition, MS4A4A+ macrophages that exhibit M2-phenotype were enriched in the tumoral niche and high expression of MS4A4A+was associated with poor prognosis in the cohort data. The ligand-receptor-based intercellular communication analysis revealed the tight interaction of MUC1+ malignant cells and ZEB1+ endothelial cells, providing mechanistic information for MCA angiogenesis and molecular targets for subsequent translational applications. Our study provides novel insights into communications among tumour cells with stromal and immune cells that are significantly enriched in the TME during MCA progression, presenting potential prognostic biomarkers and therapeutic strategies for MCA. Tumour microenvironment profiling of MCA is developed. MUC1+ tumour cells interplay with FGF7+/THBS1+ myofibroblasts to promote MCA development. MS4A4A+ macrophages exhibit M2 phenotype in MCA. ZEB1+ endotheliocytes engage in EndMT process in MCA.

Association of Electrospinning and 3D Bioprinting techniques for fabrication of cellularized vascular grafts: a proof of concept

Association of Electrospinning and 3D Bioprinting techniques for fabrication of cellularized vascular grafts: a proof of concept

Applications of 3D Bioprinting Technology to Brain Cells and Brain Tumor Models: Special Emphasis to Glioblastoma.

Primary brain tumor is one of the most fatal diseases. The most malignant type among them, glioblastoma (GBM), has low survival rates. Standard treatments reduce the life quality of patients due to serious side effects. Tumor aggressiveness and the unique structure of the brain render the removal of tumors and the development of new therapies challenging. To elucidate the characteristics of brain tumors and examine their response to drugs, realistic systems that mimic the tumor environment and cellular crosstalk are desperately needed. In the past decade, 3D GBM models have been presented as excellent platforms as they allowed the investigation of the phenotypes of GBM and testing innovative therapeutic strategies. In that scope, 3D bioprinting technology offers utilities such as fabricating realistic 3D bioprinted structures in a layer-by-layer manner and precisely controlled deposition of materials and cells, and they can be integrated with other technologies like the microfluidics approach. This Review covers studies that investigated 3D bioprinted brain tumor models, especially GBM using 3D bioprinting techniques and essential parameters that affect the result and quality of the study like frequently used cells, the type and physical characteristics of hydrogel, bioprinting conditions, cross-linking methods, and characterization techniques.

Bioprinting of a Liposomal Oxygen-Releasing Scaffold for Ovary Tissue Engineering.

This study addresses a critical challenge in bioprinting for regenerative medicine, specifically the issue of hypoxia compromising cell viability in engineered tissues. To overcome this hurdle, a novel approach using a microfluidic bioprinter is used to create a two-layer structure resembling the human ovary. This structure incorporates a liposomal oxygen-releasing system to enhance cell viability. The bioprinting technique enables the simultaneous extrusion of two distinct bioinks, namely, bioink A (comprising alginate 1% and 5 mg/mL PEGylated fibrinogen in a 20:1 molar ratio) and bioink B (containing alginate 0.5%). In addition, liposomal catalase and hydrogen peroxide (H2O2) are synthesized and incorporated into bioinks A and B, respectively. The liposomes are prepared using thin film hydration with a monodisperse size (140-160 nm) and high encapsulation efficiency. To assess construct functionality, isolated human ovarian cells are added to bioink A. The bioprinted constructs, with or without liposomal oxygen-releasing systems, are cultured under hypoxic and normoxic conditions for 3 days. Live/Dead assay results demonstrate that liposomal oxygen-releasing systems effectively preserve cell viability in hypoxic conditions, resembling viability under normoxic conditions without liposomes. PrestoBlue assay reveals significantly higher mitochondrial activity in constructs with liposomal oxygen delivery systems under both hypoxic and normoxic conditions. The evaluation of apoptosis status through annexin V immunostaining shows that liposomal oxygen-releasing scaffolds successfully protect cells from hypoxic stress, exhibiting a proportion of apoptotic cells similar to normoxic conditions. In contrast, constructs lacking liposomes in hypoxic conditions exhibit a higher incidence of cells in early-stage apoptosis. In conclusion, the study demonstrates the promising potential of bioprinted oxygen-releasing liposomal scaffolds to protect ovarian stromal cells in hypoxic environments. These innovative scaffolds not only offer protection but also recapitulate the mechanical differences between the medulla and the cortex in the normal ovary structure. This opens new avenues for advanced ovary tissue engineering and transplantation strategies.

In Situ Bioprinting: Process, Bioinks, and Applications.

Traditional tissue engineering methods face challenges, such as fabrication, implantation of irregularly shaped scaffolds, and limited accessibility for immediate healthcare providers. In situ bioprinting, an alternate strategy, involves direct deposition of biomaterials, cells, and bioactive factors at the site, facilitating on-site fabrication of intricate tissue, which can offer a patient-specific personalized approach and align with the principles of precision medicine. It can be applied using a handled device and robotic arms to various tissues, including skin, bone, cartilage, muscle, and composite tissues. Bioinks, the critical components of bioprinting that support cell viability and tissue development, play a crucial role in the success of in situ bioprinting. This review discusses in situ bioprinting techniques, the materials used for bioinks, and their critical properties for successful applications. Finally, we discuss the challenges and future trends in accelerating in situ printing to translate this technology in a clinical settings for personalized regenerative medicine.

Construction Form and Application of Three-Dimensional Bioprinting Ink Containing Hydroxyapatite.

With the increasing prevalence of bone tissue diseases, three-dimensional (3D) bioprinting applied to bone tissue engineering for treatment has received a lot of interests in recent years. The research and popularization of 3D bioprinting in bone tissue engineering require bioinks with good performance, which is closely related to ideal material and appropriate construction form. Hydroxyapatite (HAp) is the inorganic component of natural bone and has been widely used in bone tissue engineering and other fields due to its good biological and physicochemical properties. Previous studies have prepared different bioinks containing HAp and evaluated their properties in various aspects. Most bioinks showed significant improvement in terms of rheology and biocompatibility; however, not all of them had sufficiently favorable mechanical properties and antimicrobial activity. The deficiencies in properties of bioink and 3D bioprinting technology limited the applications of bioinks containing HAp in clinical trials. This review article summarizes the construction forms of bioinks containing HAp and its modifications in previous studies, as well as the 3D bioprinting techniques adopted to print bioink containing HAp. In addition, this article summarizes the advantages and underlying mechanisms of bioink containing HAp, as well as its limitations, and suggests possible improvement to facilitate the development of bone tissue engineering bioinks containing HAp in the future.

The development of a modular and open-source multi-head 3D bioprinter for fabricating complex structures

Various 3D bioprinting techniques have been introduced and developed to fabricate biomimetic constructs based on biomaterials or cell-laden bioinks to create functionally engineered tissues or organs for tissue engineering applications. However, single-biomaterial printing techniques often fail to replicate the intricate compositions and diversity found in native tissues. Multi-bioinks or multi-biomaterials in bioprinting can be utilized through either a single printhead or multiple separate printheads. However, the cost of commercially available multi-heads for bioprinting is prohibitively high, hindering their application in tissue engineering endeavors. Additionally, each bioink or biomaterial possesses unique printing characteristics that are best suited for specific printing techniques. The current study presents the development of a modular and cost-effective dual-head position bioprinter based on an open-source approach using Marlin firmware. The highlighted features of the 3D bioprinter include the use of various power sources such as compressed air and electricity for the printheads, the integration of a movable printhead mechanism with a wiper arm to prevent collisions with large printed samples during printing, a printhead adapter, as well as nozzle kits designed in a modular form for easy replacement for specific bio-applications. Therefore, despite the presence of two positions to mount the printheads, the custom-designed bioprinter exhibits the capability to flexibly accommodate four distinct printhead modules and three modular nozzle kits to print various biomaterials, such as polycaprolactone (PCL) and its composites with sodium alginate (SA), tricalcium phosphate (TCP) and hydrogel mixtures including SA, gelatin (GL), and k-carrageenan (κ-Carr). Complex tissue scaffolds were successfully fabricated using multi-biomaterials to showcase the versatility of the bioprinter, thereby demonstrating its potential for a wide range of tissue engineering applications.

3D Bioprinting of Multicellular Stem Cell‐Derived Constructs to Model Pancreatic Cell Differentiation

AbstractIn vitro models of the pancreas can aid in developing therapies for pancreatic diseases. Nonetheless, most pancreatic tissue engineering is limited to insulin‐secreting β‐cells or pancreatic adenocarcinoma models. Combining all essential tissue components, including exocrine, endocrine, and blood vasculature, is crucial to recapitulate native tissue organization. In this study, extrusion‐based 3D bioprinting to create pancreatic tissue constructs containing both endocrine and exocrine compartments is exploited. Mouse pluripotent stem cell‐derived pancreatic progenitors, pancreatic endothelial cells, and mesenchymal stem cells are bioprinted. During postprinting cultivation, the cells differentiated into exocrine and endocrine lineages, resulting in vascularized pancreatic tissue‐like constructs with multiple cell types. However, the bioprinted constructs contracted significantly postprinting, hindering control of cell positioning and shape preservation. Therefore, 2 strategies to reduce the contraction and deformation of the bioprinted constructs are developed. These bioprinting techniques and biomaterial combinations allow us to investigate the influence of construct design and cellular composition on pancreatic cell fate. The results reveal that increased construct stiffness and endothelial component presence significantly promoted endocrine while suppressing exocrine differentiation. Overall, a novel strategy for pancreatic tissue engineering that advances and holds promise for pancreas disease and development modeling, as well as pharmaceutical testing is demonstrated.