Search

Formation of graded biomaterials to render shape-morphing scaffolds for 4D biofabrication holds great promise in fabrication of complex structures and the recapitulation of critical dynamics for tissue/organ regeneration. Here we describe a facile generation of an adjustable and robust gradient using a single- or multi-material one-step fabrication strategy for 4D biofabrication. By simply photocrosslinking a mixed solution of a photocrosslinkable polymer macromer, photoinitiator (PI), UV absorber and live cells, a cell-laden gradient hydrogel with pre-programmable deformation can be generated. Gradient formation was demonstrated in various polymers including poly(ethylene glycol) (PEG), alginate, and gelatin derivatives using various UV absorbers that present overlap in UV spectrum with that of the PI UV absorbance spectrum. Moreover, this simple and effective method was used as a universal platform to integrate with other hydrogel-engineering techniques such as photomask-aided microfabrication, photo-patterning, ion-transfer printing, and 3D bioprinting to fabricate more advanced cell-laden scaffold structures. Lastly, proof-of-concept 4D tissue engineering was demonstrated in a study of 4D bone-like tissue formation. The strategy's simplicity along with its versatility paves a new way in solving the hurdle of achieving temporal shape changes in cell-laden single-component hydrogel scaffolds and may expedite the development of 4D biofabricated constructs for biological applications., Graphical abstract Image 1, Highlights • A one-material one-step gradient method for 4D hydrogel biofabrication was developed. • The gradient range and orientation within the hydrogels can be easily adjusted. • The strategy was demonstrated to be effective in a variety of hydrogel systems. • The system is highly compatible with a variety of other biofabrication technologies. • With this system, 4D bone-like tissue formation was demonstrated.

Regeneration of osteochondral tissue is of great potentialities in repairing severe osteochondral defects. However, anisotropic physiological characteristics and tissue linage difference make the regeneration of osteochondral tissue remain a huge challenge. Herein, a multicellular system based on a bilayered co-culture scaffold mimicking osteochondral tissues was successfully developed for an alternative of osteochondral regeneration via a 3D bioprinting strategy. The dual function of integrally repairing both cartilage and bone could be achieved by designing multiple-cells distribution and a cell-induced bioink containing bioceramic particles. As an important bioactive agent, the Li-Mg-Si bioceramics-containing bioink exhibited the function of simultaneously stimulating multiple cells for differentiation towards specific directions. The 3D bioprinted co-culture scaffolds showed the capacity for osteochondral tissue regeneration by inducing osteogenic and chondrogenic differentiation in vitro and accelerating the repair of severe osteochondral defects in vivo. This study offers a potential strategy for complex tissue reconstruction through bioprinting multiple tissue cells in combination of bioceramics-stimulating bioinks.

Three-dimensional (3D) extrusion-based bioprinting is widely used in tissue engineering and regenerative medicine to create cell-incorporated constructs or scaffolds based on the extrusion technique. One critical issue in 3D extrusion-based bioprinting is printability or the capability to form and maintain reproducible 3D scaffolds from bioink (a mixture of biomaterials and cells). Research shows that printability can be affected by many factors or parameters, including those associated with the bioink, printing process, and scaffold design, but these are far from certain. This review highlights recent developments in the printability assessment of extrusion-based bioprinting with a focus on the definition of printability, printability measurements and characterization, and printability-affecting factors. Key issues and challenges related to printability are also identified and discussed, along with approaches or strategies for improving printability in extrusion-based bioprinting., Graphical abstract Image 1, Highlights • Focusing on one of the critical challenges in 3D bioprinting called “printability”. • Investigating factors, including those associated with bioink, printing process, and scaffold design affecting printability. • Highlights the recent development in the discovery of printability for the extrusion bioprinting. • Providing a systematic review on “how printability is measured and characterized”. • Identifying key challenges in the printability discovery.

Various cell aggregate culture technologies have been developed and actively applied to tissue engineering and organ-on-a-chip. However, the conventional culture technologies are labor-intensive, and their outcomes are highly user dependent. In addition, the technologies cannot be used to produce three-dimensional (3D) complex tissues. In this regard, 3D cell aggregate printing technology has attracted increased attention from many researchers owing to its 3D processability. The technology allows the fabrication of 3D freeform constructs using multiple types of cell aggregates in an automated manner. Technological advancement has resulted in the development of a printing technology with a high resolution of approximately 20 μm in 3D space. A high-speed printing technology that can print a cell aggregate in milliseconds has also been introduced. The developed aggregate printing technologies are being actively applied to produce various types of engineered tissues. Although various types of high-performance printing technologies have been developed, there are still some technical obstacles in the fabrication of engineered tissues that mimic the structure and function of native tissues. This review highlights the central importance and current technical level of 3D cell aggregate printing technology, and their applications to tissue/disease models, artificial tissues, and drug-screening platforms. The paper also discusses the remaining hurdles and future directions of the printing processes.

Cancer models are essential in cancer research and for new drug development pipelines. However, conventional cancer tissue models have failed to capture the human cancer physiology, thus hindering drug discovery. The major challenge is the establishment of physiologically relevant cancer models that reflect the complexity of the tumor microenvironment (TME). The TME is a highly complex milieu composed of diverse factors that are associated with cancer progression and metastasis, as well as with the development of cancer resistance to therapeutics. To emulate the TME, 3D bioprinting has emerged as a way to create engineered cancer tissue models. Bioprinted cancer tissue models have the potential to recapitulate cancer pathology and increased drug resistance in an organ-mimicking 3D environment. This review overviews the bioprinting technologies used for the engineering of cancer tissue models and provides a future perspective on bioprinting to further advance cancer research.

Existing methods of engineering alternatives to restore or replace damaged or lost tissues are not satisfactory due to the lack of suitable constructs that can fit precisely, function properly and integrate into host tissues. Recently, three-dimensional (3D) bioprinting approaches have been developed to enable the fabrication of pre-programmed synthetic tissue constructs that have precise geometries and controlled cellular composition and spatial distribution. New bioinks with electroconductive properties have the potential to influence cellular fates and function for directed healing of different tissue types including bone, heart and nervous tissue with the possibility of improved outcomes. In the present paper, we review the use of electroconductive biomaterials for the engineering of tissues via 3D printing and 3D bioprinting. Despite significant advances, there remain challenges to effective tissue replacement and we address these challenges and describe new approaches to advanced tissue engineering.

Clinically, the treatment of bone defects remains a significant challenge, as it requires autogenous bone grafts or bone graft substitutes. However, the existing biomaterials often fail to meet the clinical requirements in terms of structural support, bone induction, and controllable biodegradability. In the treatment of bone defects, 3D porous scaffolds have attracted much attention in the orthopedic field. In terms of appearance and microstructure, complex bone scaffolds created by 3D printing technology are similar to human bone. On this basis, the combination of active substances, including cells and growth factors, is more conducive to bone tissue reconstruction, which is of great significance for the personalized treatment of bone defects. With the continuous development of 3D printing technology, it has been widely used in bone defect repair as well as diagnosis and rehabilitation, creating an emerging industry with excellent market potential. Meanwhile, the diverse combination of 3D printing technology with multi-disciplinary fields, such as tissue engineering, digital medicine, and materials science, has made 3D printing products with good biocompatibility, excellent osteoinductive capacity, and stable mechanical properties. In the clinical application of the repair of bone defects, various biological materials and 3D printing methods have emerged to make patient-specific bioactive scaffolds. The microstructure of 3D printed scaffolds can meet the complex needs of bone defect repair and support the personalized treatment of patients. Some of the new materials and technologies that emerged from the 3D printing industry's advent in the past decade successfully translated into clinical practice. In this article, we first introduced the development and application of different types of materials that were used in 3D bioprinting, including metal, ceramic materials, polymer materials, composite materials, and cell tissue. The combined application of 3D bioprinting and other manufacturing methods used for bone tissue engineering are also discussed in this article. Finally, we discussed the bottleneck of 3D bioprinting technique and forecasted its research orientation and prospect.

Conventional approaches in drug development involve testing on 2D-cultured mammalian cells, followed by experiments in rodents. Although this is the common strategy, it has significant drawbacks: in 2D cell culture with human cells, the cultivation at normoxic conditions on a plastic or glass surface is an artificial situation that significantly changes energy metabolism, shape and intracellular signaling, which in turn directly affects drug response. On the other hand, rodents as the most frequently used animal models have evolutionarily separated from primates about 100 million years ago, with significant differences in physiology, which frequently leads to results not reproducible in humans. As an alternative, spheroid technology and micro-organoids have evolved in the last decade to provide 3D context for cells similar to native tissue. However, organoids used for drug testing are usually just in the 50–100 micrometers range and thereby too small to mimic micro-environmental tissue conditions such as limited nutrient and oxygen availability. An attractive alternative offers 3D bioprinting as this allows fabrication of human tissue equivalents from scratch with hollow structures for perfusion and strict spatiotemporal control over the deposition of cells and extracellular matrix proteins. Thereby, tissue surrogates with defined geometry are fabricated that offer unique opportunities in exploring cellular cross-talk, mechanobiology and morphogenesis. These tissue-equivalents are also very attractive tools in drug testing, as bioprinting enables standardized production, parallelization, and application-tailored design of human tissue, of human disease models and patient-specific tissue avatars. This review, therefore, summarizes recent advances in 3D bioprinting technology and its application for drug screening.

Three-dimensional (3D) bioprinting is rapidly evolving, offering great potential for manufacturing functional tissue analogs for use in diverse biomedical applications, including regenerative medicine, drug delivery, and disease modeling. Biomaterials used as bioinks in printing processes must meet strict physiochemical and biomechanical requirements to ensure adequate printing fidelity, while closely mimicking the characteristics of the native tissue. To achieve this goal, nanomaterials are increasingly being investigated as a robust tool to functionalize bioink materials. In this review, we discuss the growing role of different nano-biomaterials in engineering functional bioinks for a variety of tissue engineering applications. The development and commercialization of these nanomaterial solutions for 3D bioprinting would be a significant step towards clinical translation of biofabrication.

Over the last few decades, the world has witnessed multiple viral pandemics, the current severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) pandemic being the worst and most devastating one, claiming millions of lives worldwide. Physicians, scientists, and engineers worldwide have joined hands in dealing with the current situation at an impressive speed and efficiency. One of the major reasons for the delay in response is our limited understanding of the mechanism of action and individual effects of the virus on different tissues and organs. Advances in 3D bioprinting have opened up a whole new area to explore and utilize the technology in fabricating models of these tissues and organs, recapitulating in vivo environment. These biomimetic models can not only be utilized in learning the infection pathways and drug toxicology studies but also minimize the need for animal models and shorten the time span for human clinical trials. The current review aims to integrate the existing developments in bioprinting techniques, and their implementation to develop tissue models, which has implications for SARS-CoV-2 infection. Future translation of these models has also been discussed with respect to the pandemic.

With the current rapidly growing global population, the animal product industry faces challenges which not only demand drastically increased amounts of animal products but also have to limit the emission of greenhouse gases and animal waste. These issues can be solved by the combination of microfabrication and tissue engineering techniques, which utilize the microtissue as a building component for larger tissue assembly to fabricate animal products. Various methods for the assembly of microtissue have been proposed such as spinning, cell layering, and 3D bioprinting to mimic the intricate morphology and function of the in vivo animal tissues. Some of the demonstrations on cultured meat and leather-like materials present promising outlooks on the emerging field of in vitro production of animal products.

Three-dimensional (3D) bioprinting based on traditional 3D printing is an emerging technology that is used to precisely assemble biocompatible materials and cells or bioactive factors into advanced tissue engineering solutions. Similar technology, particularly photo-cured bioprinting strategies, plays an important role in the field of tissue engineering research. The successful implementation of 3D bioprinting is based on the properties of photopolymerized materials. Photocrosslinkable hydrogel is an attractive biomaterial that is polymerized rapidly and enables process control in space and time. Photopolymerization is frequently initiated by ultraviolet (UV) or visible light. However, UV light may cause cell damage and thereby, affect cell viability. Thus, visible light is considered to be more biocompatible than UV light for bioprinting. In this review, we provide an overview of photo curing-based bioprinting technologies, and describe a visible light crosslinkable bioink, including its crosslinking mechanisms, types of visible light initiator, and biomedical applications. We also discuss existing challenges and prospects of visible light-induced 3D bioprinting devices and hydrogels in biomedical areas.

3D bioprinting has emerged as a promising technology in the field of tissue engineering and regenerative medicine due to its ability to create anatomically complex tissue substitutes. However, it still remains challenging to develop bioactive bioinks that provide appropriate and permissive environments to instruct and guide the regenerative process in vitro and in vivo. In this study alginate sulfate, a sulfated glycosaminoglycan (sGAG) mimic, was used to functionalize an alginate-gelatin methacryloyl (GelMA) interpenetrating network (IPN) bioink to enable the bioprinting of cartilaginous tissues. The inclusion of alginate sulfate had a limited influence on the viscosity, shear-thinning and thixotropic properties of the IPN bioink, enabling high-fidelity bioprinting and supporting mesenchymal stem cell (MSC) viability post-printing. The stiffness of printed IPN constructs greatly exceeded that achieved by printing alginate or GelMA alone, while maintaining resilience and toughness. Furthermore, given the high affinity of alginate sulfate to heparin-binding growth factors, the sulfated IPN bioink supported the sustained release of transforming growth factor-β3 (TGF-β3), providing an environment that supported robust chondrogenesis in vitro, with little evidence of hypertrophy or mineralization over extended culture periods. Such bioprinted constructs also supported chondrogenesis in vivo, with the controlled release of TGF-β3 promoting significantly higher levels of cartilage-specific extracellular matrix deposition. Altogether, these results demonstrate the potential of bioprinting sulfated bioinks as part of a 'single-stage' or 'point-of-care' strategy for regenerating cartilaginous tissues. STATEMENT OF SIGNIFICANCE: This study highlights the potential of using sulfated interpenetrating network (IPN) bioink to support the regeneration of phenotypically stable articular cartilage. Construction of interpenetrating networks in the bioink enables unique high-fidelity bioprinting and provides synergistic increases in mechanical properties. The presence of alginate sulfate enables the capacity of high affinity-binding of TGF-β3, which promoted robust chondrogenesis in vitro and in vivo.

Three-dimensional (3D) bioprinting of photo-cross-linkable hydrogel precursors has attracted great interest in various tissue engineering and drug screening applications, as the biochemical and biophysical properties of the resultant hydrogel structures can be tuned spatiotemporally to provide cells with physiologically relevant microenvironments. In particular, these bioinks benefit from great biofunctional versatility that can be designed to direct cells toward a desired behavior. Despite significant progress in the field, the 3D printing of cell-laden photo-cross-linkable bioinks with low polymer concentrations has remained a challenge, as rapidly stabilizing these bioinks and transforming them to hydrogel filaments is hindered by their low viscosity. Additionally, reaching an optimized print condition has often been challenging due to the large number of print parameters involved in 3D bioprinting setups. Therefore, computational modeling has occasionally been employed to understand the impact of various print parameters and reduce the time and resources required to determine these effects in experimental settings. Here, we report a novel 3D bioprinting strategy for fabricating hydrogel fibrous structures of gelatin methacryloyl (GelMA) with superior control over polymer concentration, particularly in a relatively low range from ∼1% (w/v) to 6% (w/v), using a microfluidic printhead. The printhead features a coaxial core-sheath flow, coupled with a photo-cross-linking system, allowing for the in situ cross-linking of GelMA and the generation of hydrogel filaments. A computational model was developed to determine the optimal ranges of process parameters and inform about the diffusive and fluid dynamic behavior of the coaxial flow. The cytocompatibility of the biofabrication system was determined via bioprinting cell-laden bioinks containing U87-MG cells. Notably, the established pipeline from computational modeling to bioprinting has great potential to be applied to a wide range of photo-cross-linkable bioinks to generate living tissues with various material and cellular characteristics.

Mammalian cells are widely used in biotechnology and regenerative medicine applications, including recombinant protein production, cell therapy, 3D bioprinting, and ex vivo engineering of tissues and organs. Unlike unicellular organisms, fungi, or plants, animal cells lack a protective cell wall, and therefore are more sensitive to processing conditions, particularly mechanical forces. Hydrodynamic forces during capillary flow can damage the plasma membrane and impact cell viability and functions, limiting the yields of protein production or the success of injection-based cell delivery and 3D bioprinting of artificial tissues and organs. Here, we present nanocoating individual murine fibroblasts as a model organism with silk-based artificial cell walls to protect against mechanical stress. Cells were coated with three bilayers of silk polyelectrolytes through layer-by-layer electrostatic deposition and subjected to mechanical stress by extrusion through needles with small inner diameters or shearing using a rheometer. The silk nanocoatings preserved membrane integrity, cell survival, and proliferation after exposure to stress in viscous polyethylene glycol solution, providing a useful strategy for cytoprotection during cell delivery and 3D bioprinting applications. Use of mammalian cells is a necessity in the industrial production of biopharmaceuticals because of proper protein folding through chaperone systems and post-translational modifications such as glycosylation being essential for functional products. Moreover, transplantation of human cells holds great promise in regeneration of serious injuries in the bone, brain, or spinal cord as well as debilitating diseases such as stroke, diabetes, arterial dysfunctions, and cancers. Cell delivery by direct injection or within 3D-printed scaffolds with well-defined, patient-specific geometry and composition has potential in filling the gap of insufficient organ transplantation. Both the delivery of mammalian cells into a bioreactor through transfer pipes or cell extrusion through fine needles or nozzles for injection-based delivery or bioprinting, however, may expose the cells to extremes of mechanical stress that impair cell viability and reduce the yield of production or overall success of the transplantation therapy. The silk nanocoatings on individual cells demonstrated in this study acted as artificial cell walls for mammalian cells and provided protection against mechanical stress during capillary flow or exposure to a constant shear force and significantly limited the loss of membrane integrity and necrotic or apoptotic cell death. The safety and simplicity of the approach involving protein-based, biocompatible silk fibroin processed through organic solvent-free, nature-friendly modification pathways allows for rapid translation in biotechnology and regenerative medicine to improve cell delivery applications.

In the last few decades, tissue engineering evolved as an exciting multidisciplinary field of research. The primary objective of this review is to critically analyze 3D bioprinting-based tissue engineering approaches for urinary bladder and urethra reconstruction. This review critically examines the in vitro and in vivo outcomes of natural, artificial, and hybrid scaffolds. The translational strategies at achieving clinically desired properties and an overview of biomechanical characteristics of urological biomaterials are discussed here. Notably, since the bladder is under continuous dynamic loading and unloading conditions, it is highlighted that 3D bioprinted scaffolds should withstand the biomechanical forces experienced by the native urological tissues and mimic the viscoelastic property of the native bladder tissue. It has been emphasized that 3D bioprinting with biomolecular hydrogel-functional bioink may be a possible solution to create tissue-engineered patient-specific grafts. The review closes with the authors’ perspective on relevant challenges associated with the clinical translation.

There has been an increasing demand for bioengineered blood vessels for utilization in both regenerative medicine and drug screening. However, the availability of a true bioengineered vascular graft remains limited. Three-dimensional (3D) bioprinting presents a potential approach for fabricating blood vessels or vascularized tissue constructs of various architectures and sizes for transplantation and regeneration. In this review, we summarize the basic biology of different blood vessels, as well as 3D bioprinting approaches and bioink designs that have been applied to fabricate vascular and vascularized tissue constructs, with a focus on small-diameter blood vessels.

Tissue engineering technology provides effective alternative treatments for tracheal reconstruction. The formation of a functional microvascular network is essential to support cell metabolism and ensure the long-term survival of grafts. Although several tracheal replacement therapy strategies have been developed in the past, the critical significance of the formation of microvascular networks in 3D scaffolds has not attracted sufficient attention. Here, we review key technologies and related factors of microvascular network construction in tissue-engineered trachea and explore optimized preparation processes of vascularized functional tissues for clinical applications.

Appropriate biomimetic scaffolds created via 3D bioprinting are promising methods for treating damaged menisci. However, given the unique anatomical structure and complex stress environment of the meniscus, many studies have adopted various techniques to take full advantage of different materials, such as the printing combined with infusion, or electrospining, to chase the biomimetic meniscus, which makes the process complicated to some extent. Some researchers have tried to tackle the challenges only by 3D biopringting, while its alternative materials and models have been constrained. In this study, based on a multilayer biomimetic strategy, we optimized the preparation of meniscus-derived bioink, gelatin methacrylate (GelMA)/meniscal extracellular matrix (MECM), to take printability and cytocompatibility into account together. Subsequently, a customized 3D bioprinting system featuring a dual nozzle + multitemperature printing was used to integrate the advantages of polycaprolactone (PCL) and meniscal fibrocartilage chondrocytes (MFCs)-laden GelMA/MECM bioink to complete the biomimetic meniscal scaffold, which had the best biomimetic features in terms of morphology and components. Furthermore, cell viability, mechanics, biodegradation and tissue formation in vivo were performed to ensure that the scaffold had sufficient feasibility and functionality, thereby providing a reliable basis for its application in tissue engineering., Graphical abstract Image 1, Highlights • We have optimized the preparation of meniscus-derived bioink with good printability and cytocompatibility. • A customized printing system for biomimetic meniscus, the dual-nozzle + multitemperature printing system was developed. • We have achieved multilayer meniscal biomimetic strategy, especially the best biomimetics of morphology and components. • Focusing on application prospect, we designed a few experiments to verity the feasibility and functionality of the scaffold.

Major limitation of 3D bioprinting is the poor understanding of the role of bioink in modulating molecular signaling pathways. Phenotypically stable engineered articular cartilage was fabricated using silk fibroin–gelatin (SF-G) bioink and progenitor cells or mature articular chondrocytes. In the current study, role of SF-G bioink in modulating in vitro chondrogenic signaling pathways in human bone marrow-derived stromal cells (hMSCs) is elucidated. The interaction between SF-G bioink and hMSCs augmented several chondrogenic pathways, including Wnt, HIF-1, and Notch. We explored the debatable role of TGF-β signaling, by assessing the differential protein expression by hMSCs-laden bioprinted constructs in the presence and absence of TGF-β3. hMSCs-laden bioprinted constructs contained a large percentage of collagen type II and Filamin-B, typical to the native articular cartilage. Hypertrophy markers were not identified following TGF-β3 addition. This is first detailed proteomics analysis to identify articular cartilage-specific pathways in SF-G-based 3D bioprinted construct.

The three-dimensional (3D) marrow microenvironment plays an essential role in regulating human cord blood-derived CD34+ cells (hCB-CD34+) migration, proliferation, and differentiation. Extensive in vitro and in vivo studies have aimed to recapitulate the main components of the bone marrow (BM) niche. Nonetheless, the models are limited by a lack of heterogeneity and compound structure. Here, we fabricated coaxial extruded core-shell tubular scaffolds and extrusion-based bioprinted cell-laden mesh scaffolds to mimic the functional niche in vitro. A multicellular mesh scaffold and two different core-shell tubular scaffolds were developed with human bone marrow-derived mesenchymal stromal cells (BMSCs) in comparison with a conventional 2D coculture system. A clear cell-cell connection was established in all three bioprinted constructs. Cell distribution and morphology were observed in different systems with scanning electron microscopy (SEM). Collected hCB-CD34+ cells were characterized by various stem cell-specific and lineage-specific phenotypic parameters. The results showed that compared with hCB-CD34+ cells cocultured with BMSCs in Petri dishes, the self-renewal potential of hCB-CD34+ cells was stronger in the tubular scaffolds after 14 days. Besides, cells in these core-shell constructs tended to obtain stronger differentiation potential of lymphoid and megakaryocytes, while cells encapsulated in mesh scaffolds obtained stronger differentiation tendency into erythroid cells. Consequently, 3D bioprinting technology could partially simulate the niche of human hematopoietic stem cells. The three models have their potential in stemness maintenance and multilineage differentiation. This study can provide initial effective guidance in the directed differentiation research and related screening of drug models for hematological diseases.

Regenerative engineering is defined as the convergence of the disciplines of advanced material science, stem cell science, physics, developmental biology and clinical translation for the regeneration of complex tissues and organ systems. It is an expansion of tissue engineering, which was first developed as a method of repair and restoration of human tissue. In the past three decades, advances in regenerative engineering have made it possible to treat a variety of clinical challenges by utilizing cutting-edge technology currently available to harness the body’s healing and regenerative abilities. The emergence of new information in developmental biology, stem cell science, advanced material science and nanotechnology have provided promising concepts and approaches to regenerate complex tissues and structures.

This study reports on microstructural, mechanical, and bioactive properties of the chitosan based composite scaffolds reinforced by akermanite. Firstly, the synthesis of akermanite powders was investigated by three different methods as sol-gel, ball-milling, and spray drying, with additional heat treatments. The morphological and microstructural characterizations revealed that the akermanite powders with the homogeneous and narrow particle size distributions were successfully obtained by spray drying and following heat treatment methods. Secondly, the chitosan/akermanite composite scaffolds were produced by the combination of 3D-bioprinting and lyophilization methods. SEM analyses of produced composite scaffolds indicated the interconnected and open-pore structures. The mechanical test results showed that the increasing akermanite addition enhanced the strength of composite scaffolds significantly. The bioactivity test results presented that the composite scaffolds had good in vitro bioactivity based on their good apatite-formation abilities on their surfaces and in simulated body fluid. According to the results, the bioactive 3D-printed scaffold with improved mechanical properties promises the good potential for bone tissue engineering applications.

The tissue engineering of hard organs and tissues containing cartilage, teeth, and bones is a widely used and rapidly progressing field. One of the main features of hard organs and tissues is the mineralization of their extracellular matrices (ECM) to enable them to withstand pressure and weight. Recently, a variety of printing strategies have been developed to facilitate hard organ and tissue regeneration. Fundamentals in three-dimensional (3D) printing techniques are rapid prototyping, additive manufacturing, and layered built-up and solid-free construction. This strategy promises to replicate the multifaceted architecture of natural tissues. Nowadays, 3D bioprinting techniques have proved their potential applications in tissue engineering to construct transplantable hard organs/tissues including bone and cartilage. Though, 3D bioprinting methods still have some uncertainties to fabricate 3D hard organs/tissues. In the present review, most advanced technical improvements, experiments, and future outlooks of hard tissue engineering are discussed, as well as their relevant additive manufacturing techniques.

The bioprinting technique with specialized tissue production allows the study of biological, physiological, and behavioral changes of cancerous and non-cancerous tissues in response to pharmacological compounds in personalized medicine. To this end, to evaluate the efficacy of anticancer drugs before entering the clinical setting, tissue engineered 3D scaffolds containing breast cancer and derived from the especially patient, similar to the original tissue architecture, can potentially be used. Despite recent advances in the manufacturing of 3D bioprinted breast cancer tissue (BCT), many studies still suffer from reproducibility primarily because of the uncertainty of the materials used in the scaffolds and lack of printing methods. In this review, we present an overview of the breast cancer environment to optimize personalized treatment by examining and identifying the physiological and biological factors that mimic BCT. We also surveyed the materials and techniques related to 3D bioprinting, i.e, 3D bioprinting systems, current strategies for fabrication of 3D bioprinting tissues, cell adhesion and migration in 3D bioprinted BCT, and 3D bioprinted breast cancer metastasis models. Finally, we emphasized on the prospective future applications of 3D bioprinted cancer models for rapid and accurate drug screening in breast cancer.

For 3D bioprinted tissues to be scaled-up to clinically relevant sizes, effective prevascularisation strategies are required to provide the necessary nutrients for normal metabolism and to remove associated waste by-products. The aim of this study was to develop a bioprinting strategy to engineer prevascularised tissues in vitro and to investigate the capacity of such constructs to enhance the vascularisation and regeneration of large bone defects in vivo. From a screen of different bioinks, a fibrin-based hydrogel was found to best support human umbilical vein endothelial cell (HUVEC) sprouting and the establishment of a microvessel network. When this bioink was combined with HUVECs and supporting human bone marrow stem/stromal cells (hBMSCs), these microvessel networks persisted in vitro. Furthermore, only bioprinted tissues containing both HUVECs and hBMSCs, that were first allowed to mature in vitro, supported robust blood vessel development in vivo. To assess the therapeutic utility of this bioprinting strategy, these bioinks were used to prevascularise 3D printed polycaprolactone (PCL) scaffolds, which were subsequently implanted into critically-sized femoral bone defects in rats. Micro-computed tomography (µCT) angiography revealed increased levels of vascularisation in vivo, which correlated with higher levels of new bone formation. Such prevascularised constructs could be used to enhance the vascularisation of a range of large tissue defects, forming the basis of multiple new bioprinted therapeutics. STATEMENT OF SIGNIFICANCE: This paper demonstrates a versatile 3D bioprinting technique to improve the vascularisation of tissue engineered constructs and further demonstrates how this method can be incorporated into a bone tissue engineering strategy to improve vascularisation in a rat femoral defect model.

Hydrogels are cross-linked polymeric structures, which consist of up to approximately 90% water, the remainder is polymer chain. Retention of large volumes of water in the intermolecular space is related to the presence of hydrophilic functional groups in the network. The unique hydrogels properties, such as porosity, and biological and mechanical properties, make them suitable for a wide range of applications, especially in the medical sector. Furthermore, ease of modification and good printability are expected in 3D bioprinting technologies. Nevertheless, to maintain their structure and softness, hydrogels must be stored in suitable conditions to prevent water vaporization. The water removal from the hydrogel network results in weight reduction, structural and volumetric changes. It is a considerable challenge for the printouts manufactured by 3D bioprinting technology, where hydrogel products are exposed to drying during the production process, which may affect their shape change and shrinkage. The paper presents a crosslinking process of a hydrogel-based on sodium alginate and the shrinkage of dried hydrogels depending on the crosslinking procedure. An investigation focused on the alginate hydrogel water content, as well as shrinkage of alginate hydrogel degree depending on the concentration of the cross-linking (CaCl2) solution and the duration of the process. For longer cross-linking time or using higher cross-linking agent concentration, the cross-linking was more efficient. However, it is necessary to optimize the parameters for the bioprinting process.

Three-dimensional (3D) bioprinting is a technology under active study for use in tissue engineering and regenerative medicine. Bioink comprises cells and polymers and is the essential material for 3D bioprinting. The characteristics of the bioink affect its printability, gelation behavior, and cell compatibility. In this study, alginate derivatives were synthesized to induce rapid gelation, and a bioink was prepared by mixing these alginate derivatives with silk fibroin to enhance cell compatibility. A low-concentration (3 wt %) alginate/silk fibroin (Alg/SF) bioink was pregelated by the ionic cross-linking of Alg to increase the viscosity for 3D printing. The rheological and mechanical properties were analyzed using a rheometer and a texture meter, respectively. Analysis of cell viability and proliferation using fibroblasts (NIH-3T3) in the bioinks showed that the Alg/SF bioink has improved cytocompatibility compared to that of conventional Alg bioinks, making it a promising material for tissue engineering.

Photopolymerization is an effective method to covalently cross-link polymer chains that can be shaped into several biomedical products and devices. Additionally, polymerization reaction may induce a fluid-solid phase transformation under physiological conditions and is ideal for in vivo cross-linking of injectable polymers. The photoinitiator is a key ingredient able to absorb the energy at a specific light wavelength and create radicals that convert the liquid monomer solution into polymers. The combination of photopolymerizable polymers, containing appropriate photoinitiators, and effective curing based on dedicated light sources offers the possibility to implement photopolymerization technology in 3D bioprinting systems. Hence, cell-laden structures with high cell viability and proliferation, high accuracy in production, and good control of scaffold geometry can be biofabricated. In this review, we provide an overview of photopolymerization technology, focusing our efforts on natural polymers, the chemistry involved, and their combination with appropriate photoinitiators to be used within 3D bioprinting and manufacturing of biomedical devices. The reviewed articles showed the impact of different factors that influence the success of the photopolymerization process and the final properties of the cross-linked materials.

3D Bioprinting is a dynamically developing technology for tissue engineering and regenerative medicine. The main advantage of this technique is its ability to reproduce a given scaffold geometry and structure both in terms of the shape of the tissue-engineered construct and the distribution of its components. The key factor in bioprinting is bio ink, a cell-laden biocompatible material that mimics extracellular matrix. To meet all the requirements, the bio ink must include not only the main material, but also other components ensuring cell proliferation, differentiation and scaffold performance as a whole. The purpose of this review is to describe the most common materials applicable in bioprinting, consider their properties, prospects and limitations in cartilage restoration.

Цель исследования — определение свойств современных биоактивных композитных материалов, имеющих наибольшее преимущество для использования в травматологии и ортопедии, в том числе в хирургии позвоночника. Материал и методы. Выполнен поиск и анализ литературных источников, опубликованныех в научной базе PubMed, а также научной электронной библиотеки eLIBRARY и поисковой системе Semantic Scholar. Для поиска использованы ключевые слова: имплантаты, современные биоматериалы, композиты, тканевая инженерия, скаффолды, графен, гидрогели, 3D-биопечать, ортопедия. Нами был произведен поиск научных публикаций за период с 2010 по 2020 г. Оценивались следующие свойства: биотолерантность, биоактивность, остеокондуктив- ность, остеоиндуктивность, остеостимуляция, механическая прочность. Результаты. Созданию композитов уделяется особое внимание. Композиты изготовлены путем объединения двух или более материалов для достижения биохимических и биомеханических свойств. В производстве композитов определенное место занимает технология 3D-биопечати, благодаря которой возможна разработка индивидуального имплантата согласно заданной ситуации. Заключение. Сочетание свойств композитных материалов, указывающих на их биоактивность и прочность, а также использование 3D-технологий для формирования геометрических размеров имплантатов из них обеспечивают высокий потенциал для применения в области травматологии и ортопедии, в том числе для использования в хирургии позвоночника.

Made available in DSpace on 2021-06-26T06:17:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-04-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Scaffolds are very important element for bone regeneration issues. On this way, the purpose of this paper is the design and manufacturing of porous scaffolds fabricated by 3D printing technology from biodegradable thermoplastic polymers and calcium phosphates (in micrometric scale). So, the main aim of this research is to obtain complex porous 3D structures that present adequate mechanical properties in relation to bones, through a structured interconnectivity between the pores. Based on the 3D models of the designed scaffolds, selection and preparation of the biomaterials, the process parameters were set in order to provide conditions for the scaffolds? manufacturing. Using an additive manufacturing technology of pneumatic gelling liquid extrusion, with a bioprinter that uses pneumatic distribution system for continuous extrusion of material, two models of designed scaffolds were 3D printed and characterized by mechanical compression analyses and then, evaluated by Scanning Electron Microscopy (SEM) method. Results of Linear Static Analysis (LSA) showed that the 3D designed scaffolds meet the specifications required in the literature for specific rigidity (20?141 MPa). The stress x strain curves showed that the compressive strength values of the composite biomaterial used for all tested coupons are within the values described in the literature for trabecular bone application (range from 2 to 12 MPa). In addition, the pore sizes proven by micrographs have been within the range of application for tissue engineering (20?850 ?m), as mentioned by the literature too. Also, the SEM showed the repeatability related to the interconnectivity between pores, based on homogeneous and uniform structures, regarding adhesion between layers, dimensions of pores and constant extruded filament. Thus, development and applications of the biomaterial composed by Polycaprolactone and Amorphous Calcium Phosphate (PCL + ACP) faced to the additive manufacturing method used to perform the printing of designed scaffolds, can be considered a potential and promising novelty for application in tissue engineering field. Sao Paulo State Univ, Dept Bioproc & Biomat Engn, Araraquara Jaui Rd,Km 1, BR-14800901 Araraquara, SP, Brazil Univ Fed Sao Carlos, Dept Mech Engn, Washington Luis Rd,Km 235, BR-13565905 Sao Carlos, SP, Brazil Sao Paulo State Univ, Dept Chem, Prof Francisco Degni St 55, BR-14800060 Araraquara, SP, Brazil Sao Paulo State Univ, Dept Bioproc & Biomat Engn, Araraquara Jaui Rd,Km 1, BR-14800901 Araraquara, SP, Brazil Sao Paulo State Univ, Dept Chem, Prof Francisco Degni St 55, BR-14800060 Araraquara, SP, Brazil CNPq: 314516/20182

INTRODUCTION: The use of 3D-bioprinted ovaries has been proven to be a promising technique for preserving fertility. Stereology is an accurate method to obtain quantitative 3D information and the stereological data is the basis for 3D bioprinting ovaries. METHODS: In this study, six female mice were used to acquire the ovarian tissues. One of the two paraffin-embedded ovaries of each mouse was cut into 5 µm sections, and the other was cut into 15 µm sections and then subjected to haematoxylin and eosin staining and anti-follicle stimulating hormone receptor antibody immunohistochemistry. The volume and volume fractions of ovaries were measured by the Cavalieri method. Then, the numerical densities and total numbers of ovarian granulosa cells (OGCs) and primordial, preantral and antral follicles in serial sections were estimated using design-based stereology. RESULTS: The ovarian volume was 2.50 ± 0.32 mm(3). The volume fractions of the cortex, medulla, follicles and OGCs were 86.80% ± 2.82, 13.20% ± 2.82%, 5.60% ± 0.25% and 81.19% ± 2.57%, respectively. The numerical densities of OGCs, the primordial, preantral and antral follicles were 2.11 (± 0.28) × 10(6)/mm(3), 719.57 ± 18.04/mm(3), 71.84 ± 3.93/mm(3) and 17.29 ± 3.54/mm(3), respectively. The total number of OGCs and follicles per paraffin-embedded ovary were 5.26 (± 0.09) × 10(6) and 2013.66 ± 8.16. CONCLUSIONS: The study had obtained the stereological data of the mice ovaries, which contribute to a deeper understanding of the structure of the ovaries. Meanwhile, the data will supply information for 3D bioprinting ovaries.

Purpose of Review β cell replacement via whole pancreas or islet transplantation has greatly evolved for the cure of type 1 diabetes. Both these strategies are however still affected by several limitations. Pancreas bioengineering holds the potential to overcome these hurdles aiming to repair and regenerate β cell compartment. In this review, we detail the state-of-the-art and recent progress in the bioengineering field applied to diabetes research. Recent Findings The primary target of pancreatic bioengineering is to manufacture a construct supporting insulin activity in vivo. Scaffold-base technique, 3D bioprinting, macro-devices, insulin-secreting organoids, and pancreas-on-chip represent the most promising technologies for pancreatic bioengineering. Summary There are several factors affecting the clinical application of these technologies, and studies reported so far are encouraging but need to be optimized. Nevertheless pancreas bioengineering is evolving very quickly and its combination with stem cell research developments can only accelerate this trend.

Self-healing hydrogels attract broad attention as cell/drug carriers for direct injection into damaged tissues or as bioinks for three-dimensional (3D) printing of tissue-like constructs. For application in 3D printing, the self-healing hydrogels should maintain the steady rheological properties during printing process, and be further stabilized by secondary post-printing crosslinking. Here, a chitosan self-healing hydrogel is developed for injectable hydrogel and printable ink using phenol-functionalized chitosan and dibenzaldehyde-terminated telechelic poly(ethylene glycol). Phenol functionalization of chitosan can introduce unique interaction that allows the hydrogel to possess fast gelling rate, good self-healing ability, and long-range critical gel behavior, as well as secondary visible light-crosslinking capability. The hydrogel is easily pre-formed in a syringe and extruded through a 26-gauge needle to produce a continuous and stackable filament. The cell-laden hydrogel is successfully printed into a 3D construct. Moreover, the hydrogel is developed for modular 3D printing, where hydrogel modules (LEGO-like building blocks) are individually printed and assembled into an integrated construct followed by secondary visible light-crosslinking. The versatile phenol-functionalized chitosan self-healing hydrogel will open up numerous potential applications, particularly in 3D bioprinting and modular 3D bioprinting.

Aim: Bioink is one of the essential factors in 3D bioprinting that determines the fate of cells, in our case, umbilical cord-derived mesenchymal stem cells (UMSC). The aim was to determine if the presence of the osteoinductive factors in the bioink enhances osteodifferentiation as compared with adding them postprinting and if the UMSC and endothelial cells (EC) coculture result in better osteodifferentiation. Materials & methods: Alginate-gelatin along with UMSC–EC were bioprinted using an extrusion 3D bioprinter. Results & conclusion: The UMSC–EC interaction, as well as intrinsic addition of the differentiation components in the bioink, were observed to play a vital role in increasing the osteogenic differentiation as shown by the histochemical staining, alkaline phosphatase activity and gene expression of osteogenic markers.

Drug development is a critical step in the development pipeline of pharmaceutical industry, commonly performed in traditional cell culture and animal models. Though, those models hold critical gapsin the prediction and the translation of human pharmacokinetic (PK) and pharmacodynamics (PD) parameters. The advances in tissue engineering have allowed the combination of cell biology with microengineering techniques, offering alternatives to conventional preclinical models. Organ-on-a-chips and three-dimensional (3D) bioprinting models present the potentialityof simulating the physiological and pathological microenvironment of living organs and tissues, constituting this way,more realistic models for the assessment of absorption, distribution, metabolism and excretion (ADME) of drugs. Therefore, this review will focus on lung-on-a-chip and 3D bioprinting techniques for developing lung models that can be usedfor predicting PK/PD parameters.

Three-dimensional (3D) printing techniques have revolutionized the field of tissue engineering. This is especially favorable to construct intricate tissues such as liver, as 3D printing allows for the precise delivery of biomaterials, cells and bioactive molecules in complex geometries. Bioinks made of polymers, of both natural and synthetic origin, have been very beneficial to printing soft tissues such as liver. Using polymeric bioinks, 3D hepatic structures are printed with or without cells and biomolecules, and have been used for different tissue engineering applications. In this review, with the introduction to basic 3D printing techniques, we discuss different natural and synthetic polymers including decellularized matrices that have been employed for the 3D bioprinting of hepatic structures. Finally, we focus on recent advances in polymeric bioinks for 3D hepatic printing and their applications. The studies indicate that much work has been devoted to improvising the design, stability and longevity of the printed structures. Others focus on the printing of tissue engineered hepatic structures for applications in drug screening, regenerative medicine and disease models. More attention must now be diverted to developing personalized structures and stem cell differentiation to hepatic lineage.

Bioprinting is a rapidly developing technology for the precise design and manufacture of tissues in various biological systems or organs. Coaxial extrusion bioprinting, an emergent branch, has demonstrated a strong potential to enhance bioprinting's engineering versatility. Coaxial bioprinting assists in the fabrication of complex tissue constructs, by enabling concentric deposition of biomaterials. The fabricated tissue constructs started with simple, tubular vasculature but have been substantially developed to integrate complex cell composition and self-assembly, ECM patterning, controlled release, and multi-material gradient profiles. This review article begins with a brief overview of coaxial printing history, followed by an introduction of crucial engineering components. Afterward, we review the recent progress and untapped potential in each specific organ or biological system, and demonstrate how coaxial bioprinting facilitates the creation of tissue constructs. Ultimately, we conclude that this growing technology will contribute significantly to capabilities in the fields of in vitro modeling, pharmaceutical development, and clinical regenerative medicine., Graphical abstract Image 1, Highlights • Coaxial extrusion enables concentric multi-material deposition, a broader range of materials, and inline crosslinking. • Sophisticated tissue constructs including vasculature, skeletal, organoid, and neural systems have been demonstrated. • Novel applications of coaxial bioprinting are proposed; these benefit in vitro modeling and clinical regenerative medicine.

As a 3D bioprinting technique, hydrogel stereolithography has historically been limited in its ability to capture the spatial heterogeneity that permeates mammalian tissues and dictates structure–function relationships. This limitation stems directly from the difficulty of preventing unwanted material mixing when switching between different liquid bioinks. Accordingly, we present the development, characterization, and application of a multi-material stereolithography bioprinter that provides controlled material selection, yields precise regional feature alignment, and minimizes bioink mixing. Fluorescent tracers were first used to highlight the broad design freedoms afforded by this fabrication strategy, complemented by morphometric image analysis to validate architectural fidelity. To evaluate the bioactivity of printed gels, 344SQ lung adenocarcinoma cells were printed in a 3D core/shell architecture. These cells exhibited native phenotypic behavior as evidenced by apparent proliferation and formation of spherical multicellular aggregates. Cells were also printed as pre-formed multicellular aggregates, which appropriately developed invasive protrusions in response to hTGF-β1. Finally, we constructed a simplified model of intratumoral heterogeneity with two separate sub-populations of 344SQ cells, which together grew over 14 days to form a dense regional interface. Together, these studies highlight the potential of multi-material stereolithography to probe heterotypic interactions between distinct cell types in tissue-specific microenvironments.

Mechanical stimulation of cells embedded in scaffolds is known to increase the cellular performance toward osteogenic or chondrogenic differentiation and tissue development. Three-dimensional bioplotting of magnetically deformable scaffolds enables the spatially defined distribution of magnetically inducible scaffold regions. In this study, a magnetic bioink based on alginate (alg, 3%) and methylcellulose (MC, 9%) with incorporated magnetite microparticles (25% w/w) was developed and characterized. The size and shape of particles were monitored via scanning electron microscopy and X-ray micro-computed tomography. Shear-thinning properties of the algMC ink were maintained after the addition of different concentrations of magnetite microparticles to the ink. Its viscosity proportionally increased with the added amount of magnetite, and so did the level of saturation magnetization as determined via vibrating sample magnetometry. The printability and shape fidelity of various shapes were evaluated, so that the final composition of algMC + 25% w/w magnetite was chosen. With application of this ink, cytocompatibility was proven in indirect cell culture and bioplotting experiments using a human mesenchymal stem cell line. Toward the deformation of cell-laden scaffolds to support cell differentiation in the future, radiography allowed the real-time monitoring of magnetically induced deformation of scaffolds of different pore architectures and scaffold orientations inside the magnetic field. Varying the strand distance and scaffold design will allow fine-tuning the degree of deformation in stimulatory experiments.

Three-dimensional (3D) bioprinting is a technology that can precisely fabricate customized tissues and organs. Hydrogel materials that can embed living cells for use in 3D printing are called bioinks. However, there are only limited options of bioinks currently because they require the following features at once, such as printability, repetitive layer-by-layer stacking (stackability), structure stabilization, and biological properties. A polyurethane-gelatin double network hydrogel bioink was previously reported to own tunable modulus through changing the solid content, but cell viability at the high solid content is inevitably reduced. In the present study, the reinforcement effects of a metal-organic framework (MOF), zeolitic imidazolate framework-8 (ZIF-8), in the PUG bioink were evaluated. The printability, stackability, thermoresponsiveness, and shear-thinning behavior of the PUG-ZIF-8 composite hydrogels were examined. It was found that the PUG composite hydrogel containing 1250 μg/mL ZIF-8 crystals showed significant structural stability and modulus enhancement (∼2.5-fold). However, the PUG bioink containing 1250 μg/mL ZIF-8 crystals may lead to cell senescence or death. The cytocompatible concentration of ZIF-8 crystals in the bioink was about 875 μg/mL, and this concentration was much higher than the reported tolerable amount (∼50 μg/mL) of ZIF-8 for biomedical applications. The strong reinforcement effect of ZIF-8 and the drug-loading/sensing possibilities of MOFs may open new opportunities for using MOFs in 3D bioprinting applications.

3D bioprinting allows the production of patient-specific tissue constructs with desired structural characteristics such as high resolution, controlled swelling degree, and controlled degradation behavior by mostly using hydrogels. Crosslinking of hydrogels is an essential parameter in bioprinting applications, which is beneficial for tuning structural specifications. In this study, gelatin-alginate-whey protein isolate based hydrogels have been used for 3D printing structures in a layer-by-layer fashion. These structures were cross-linked by the Amino Acid (monomer) Decorated and Light Underpinning Conjugation Approach (ANADOLUCA) method, which is a unique, non-invasive photosensitive cross-linking technique for protein-based mixtures. In that aim, hydrogel properties (e.g., printability, biocompatibility, rheologic and mechanical behavior) and cross-linking properties (e.g., swelling and degradation behavior) were studied. Results were compared with UV and ionic cross-linking techniques, which are the abundantly used techniques in such studies. The results showed that the ANADOLUCA method can be used for in situ cross-linking under mild conditions for the printing of bio-inks, and the proposed method can be used as an alternative for UV-based and chemical cross-linking techniques.

We have developed an in situ bioprinting method that allows the printing of cells under true physiological conditions by applying self-assembling ultrashort peptides as bioinks. This method avoids cell stressing methods, such as UV-treatment, chemical crosslinking and viscous bioink printing methods. We further demonstrate that different nanomaterials can easily be synthesized or incorporated in the 3D bioprinted peptide scaffolds which opens up the possibility of functionalized 3D scaffolds.

Hydrogels based on cellulose nanofibrils (CNFs) have been widely used as scaffolds for biomedical applications, however, the poor mechanical properties of CNF hydrogels limit their use as ink for 3D bioprinting in order to generate scaffolds for tissue engineering applications. In this study, a dual crosslinkable hydrogel ink composed of a poly(ethylene glycol) (PEG) star polymer and 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO)-oxidized nanocellulose fibers (CNFs) is presented. As the resulting hydrogel had low structural integrity, at first crosslinking of CNFs was introduced by Ca2+. Strong physical interactions between CNFs and Ca2+ cations allowed easy regulation of the viscosity of the inks for extrusion printing raising the solution viscosity by more than 1.5 times depending on the amount of Ca2+ added. The resulting hydrogel had high structural integrity and was further stabilized in a second step by photo crosslinking of PEG under visible light. In only a few seconds, hydrogels with Young's modulus between ∼10 and 30 kPa were obtained just by altering the CNF and Ca2+ content. 3D printed hydrogels supported fibroblasts with excellent cell viability and proliferation. The dual crosslinkable hydrogel ink herein developed is versatile, easy to prepare, and suitable for 3D printing of bioscaffolds with highly tailored viscoelastic and mechanical properties applicable in a wide range of regenerative medicines.

Acute injury of the articular cartilage can lead to chronic disabling conditions because of the limited self-repair capability of the cartilage. Implantation of stem cells at the injury site is a viable treatment, but requires a scaffold with a precisely controlled geometry and porosity in the 3D space, high biocompatibility, and the capability of promoting chondrogenic differentiation of the implanted stem cells. Here we report the development of gelatin/hydroxyapatite (HAP) hybrid materials by microextrusion 3D bioprinting and enzymatic cross-linking as the scaffold for human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs). The scaffold supports the adhesion, growth, and proliferation of hUCB-MSCs and induces their chondrogenic differentiation in vitro. Doping HAP in the gelatin scaffold increases the fluidity of the hydrogel, improves the gelation kinetics and the rheological properties, and allows better control over 3D printing. Implanting the hUCB-MSC-laden scaffold at the injury site of the articular cartilage effectively repairs the cartilage defects in a pig model. Altogether, this work demonstrates the 3D printing of gelatin-based scaffold materials for hUCB-MSCs to repair cartilage defects as a potential treatment of articular cartilage injury.

Nerve conduits have been identified as one of the most promising treatments for peripheral nerve injuries, yet it remains unsolved how to develop ideal nerve conduits with both appropriate biological and mechanical properties. Existing nerve conduits must make trade-offs between mechanical strength and biocompatibility. Here, we propose a multi-nozzle additive-lathe 3D bioprinting technology to fabricate a bilayered nerve conduit. The materials for printing consisted of gelatin methacrylate (GelMA)-based inner layer, which was cellularized with bone marrow mesenchymal stem cells (BMSCs) and GelMA/poly(ethylene glycol) diacrylate (PEGDA)-based outer layer. The high viability and extensive morphological spreading of BMSCs encapsulated in the inner layer was achieved by adjusting the degree of methacryloyl substitution and the concentration of GelMA. Strong mechanical performance of the outer layer was obtained by the addition of PEGDA. The performance of the bilayered nerve conduits was assessed using in vitro culture of PC12 cells. The cell density of PC12 cells attached to cellularized bilayered nerve conduits was more than 4 times of that on acellular bilayered nerve conduits. The proliferation rate of PC12 cells attached to cellularized bilayered nerve conduits was over 9 times higher than that on acellular bilayered nerve conduits. These results demonstrate the additive-lathe 3D bioprinting of BMSCs embedded bilayered nerve conduits holds great potential in facilitating peripheral nerve repair., Graphical abstract Image 1, Highlights • A multi-nozzle additive-lathe 3D bioprinting technology is developed to fabricate a bilayered nerve conduit. • The outer layer of nerve conduit provide a strong mechanical property and the inner layer has a good biocompatibility. • Bone marrow mesenchymal stem cells (BMSCs) are incorporated in the inner layer of nerve conduit using bioprinting. • In vitro culture of PC12 cells demonstrates the neuron outgrowth is significantly improved in BMSCs embedded nerve conduits.

Regeneration of Intervertebral disc (IVD) is a scientific challenge because of the complex structure and composition of tissue, as well as the difficulty in achieving bionic function. Here, an anatomically correct IVD scaffold composed of biomaterials, cells, and growth factors were fabricated via three-dimensional (3D) bioprinting technology. Connective tissue growth factor (CTGF) and transforming growth factor-β3 (TGF-β3) were loaded onto polydopamine nanoparticles, which were mixed with bone marrow mesenchymal stem cells (BMSCs) for regenerating and simulating the structure and function of the nucleus pulposus and annular fibrosus. In vitro experiments confirmed that CTGF and TGF-β3 could be released from the IVD scaffold in a spatially controlled manner, and induced the corresponding BMSCs to differentiate into nucleus pulposus like cells and annulus fibrosus like cells. Next, the fabricated IVD scaffold was implanted into the dorsum subcutaneous of nude mice. The reconstructed IVD exhibited a zone-specific matrix that displayed the corresponding histological and immunological phenotypes: primarily type II collagen and glycosaminoglycan in the core zone, and type I collagen in the surrounding zone. The testing results demonstrated that it exhibited good biomechanical function of the reconstructed IVD. The results presented herein reveal the clinical application potential of the dual growth factors-releasing IVD scaffold fabricated via 3D bioprinting. However, the evaluation in large mammal animal models needs to be further studied., Graphical abstract Image 1, Highlights • PDA NPs was used as a growth factor carrier for 3D bioprinting. • Materials, cells and growth factors were combined to fabricate the scaffold by 3D bioprinting technology. • Reconstructed IVD exhibited the same zone specific matrix as the natural tissue. • Reconstructed IVD exhibited good biomechanics.

In recent years, additive manufacturing, or in other words three-dimensional (3D) printing technology, has rapidly become one of the hot topics in the world. Among the vast majority of materials, chitosan and its hybrids with various wonderful features have played a noteworthy role in this science. There are lots of studies on the applications of chitosan and its derivatives in the biomedical field, but it is worth mentioning that one of the main differences of the present work with others is the consideration of the latest research on 3D bioprinted chitosan constructs, especially for biomedical applications. For this aim, this mini review starts with a definition and a brief history of 3D printing technology and the position and role of chitosan and its hybrids in this area. Subsequently, a critical discussion on current developments of chitosan for diverse applications especially for tissue engineering is provided.

Over the past decade, 3D bioprinting technology has progressed tremendously in the field of tissue engineering in its ability to fabricate individualized biological constructs with precise geometric designability, which offers us the capability to bridge the divergence between engineered tissue constructs and natural tissues. In this work, we first review the current widely used 3D bioprinting approaches, cells, and materials. Next, the updated applications of this technique in tissue engineering, including bone tissue, cartilage tissue, vascular grafts, skin, neural tissue, heart tissue, liver tissue and lung tissue, are briefly introduced. Then, the prominent advantages of 3D bioprinting in tissue engineering are summarized in detail: rapidly prototyping the customized structure, delivering cell-laden materials with high precision in space, and engineering with a highly controllable microenvironment. The current technical deficiencies of 3D bioprinted constructs in terms of mechanical properties and cell behaviors are afterward illustrated, as well as corresponding improvements. Finally, we conclude with future perspectives about 3D bioprinting in tissue engineering.