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Characterization of printed SMC patch in culture. The proliferation graph shows increasing number of cells over a period of time in collagen patches for three initial cell concentrations (C init ), that is, 1Â10 6 , 5Â10 6 , and 10Â10 6 cells=mL. (a) The total number of cells per square millimeter in three different initial printing concentrations were measured from day 0 to 7. Inset represents an enlarged figure of 1Â10 6 cells=mL initial cell loading density. After 7 days of culturing (C sat ), 270 AE 25, 1183 AE 236, and 2097 AE 287 cells=mm 2 were observed for 1Â10 6 , 5Â10 6 , and 10Â10 6 cells=mL, respectively. The inflection time (t inflection ) of sigmoid regression curves was 2.6 days for 5Â10 6 cells=mL and 3.2 days for 10Â10 6 cells=mL. In case of 26 AE 1.7 cells=mm 2 initial cell loading density, proliferation rate of cells showed an exponential increment. The unknown factor for cell proliferation b is a factor of each exponent and sigmoid regression functions, 0.2 for 1Â10 6 cells=mL, 1.3 for 5Â10 6 cells=mL, and 1.7 for 10Â10 6 cells=mL. (b-e) Stained SMC patch images for 1Â10 6 cells=mL concentration after day(s) in culture: day 4 culture of SMC patch stained with 4 0 ,6-diamidino-2-phenylindole (DAPI) (blue) and actin (green) under a light microscope (10Â) in (b), day 7 SMCs stained with DAPI and actin in (c), SMCs stained with DAPI (blue) at day 14 in culture in (d), SMCs stained with DAPI and connexin-43 (red) at day 14 in culture in (e). Scale bar: 100 mm. Color images available online at www.liebertonline.com=ten.
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The ability to bioengineer three-dimensional (3D) tissues is a potentially powerful approach to treat diverse diseases such as cancer, loss of tissue function, or organ failure. Traditional tissue engineering methods, however, face challenges in fabricating 3D tissue constructs that resemble the native tissue microvasculature and microarchitectures...
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... The topographic color coding of the top view of these patches reveals the cell distribution over 1-7 days for 5Â10 6 cells=mL cell printing concentration (Fig. 6a- d). The color coding indicates the cell concentration in that area (see the legend). The increased cell seeding density correlates with the increased number of cells per droplet (Fig. 7a). This characterization is crucial, since it builds the logical tie between a cell-laden hydrogel droplet and a printed 3D tissue construct. However, the proliferation rate is not linear as a function of cell density and culture time. The rates show a sigmoid tendency as a function of culture duration, which indicates that initial high ...
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... frequency. The total time becomes 10 min including the ge- lation time to build a secondary layer. This processing time indicates the high-throughput aspect of the system compared to the conventional scaffold methods that take 1-2 h to build a single patch. Cells are also observed to adhere and spread within the printed cell-laden collagen layer (Fig. 7b-e). In long-term culture, cells were observed to be viable as dem- onstrated by histological stains. During days 4 and 7, the printed cells expressed actin after the printing and culturing steps (Fig. 7b, c). Patches on the 14th day of culture ex- pressed connexin-43 (Fig. 7d, e). This marks a positive turning point for the printed ...
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... methods that take 1-2 h to build a single patch. Cells are also observed to adhere and spread within the printed cell-laden collagen layer (Fig. 7b-e). In long-term culture, cells were observed to be viable as dem- onstrated by histological stains. During days 4 and 7, the printed cells expressed actin after the printing and culturing steps (Fig. 7b, c). Patches on the 14th day of culture ex- pressed connexin-43 (Fig. 7d, e). This marks a positive turning point for the printed patches and indicates future possibilities for tissue engineering by this 3D bioprinting platform technology. This technology employed for tissue engineering and regenerative medicine could create avenues for ...
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... to adhere and spread within the printed cell-laden collagen layer (Fig. 7b-e). In long-term culture, cells were observed to be viable as dem- onstrated by histological stains. During days 4 and 7, the printed cells expressed actin after the printing and culturing steps (Fig. 7b, c). Patches on the 14th day of culture ex- pressed connexin-43 (Fig. 7d, e). This marks a positive turning point for the printed patches and indicates future possibilities for tissue engineering by this 3D bioprinting platform technology. This technology employed for tissue engineering and regenerative medicine could create avenues for functional tissues and could create a clinical impact by enhancing the ...
Citations
... Bioink fragments can cause blockages in the orifices, and consequently, the selected bioink must have a low viscosity (<10 mPa·s) and cell density (<10 6 cells/mL). 40,41 The acoustic droplet jet bioprinting process may introduce interfering factors that disrupt process control, and sound waves could fail to spray droplets of viscous bioinks with high cell concentrations. Controlled by the valve opening time of pressure, the microvalve bioprinter is more suitable for high viscous materials, but the bioprinting of highresolution bioinks is relatively poor. ...
Three-dimensional (3D) bioprinting is a promising additive manufacturing technology that uses imaging data and computer-assisted deposition of biological materials or cells to reconstruct complex 3D structures accurately. This technology has progressed rapidly, in part because of its integration across multiple disciplines and combination with other technologies for clinical applications. Advances in experimental research and clinical applications related to otorhinolaryngology have led to the development of diagnostic and treatment methods based on 3D bioprinting, including the development of tissue engineering scaffolds, biosensors, organ chips, and organoids, surgical planning, graft construction, and medical education. Additionally, otorhinolaryngologists will be better equipped to treat tissue function defects with personalized printed graft implants. It is also expected that 3D printing can be used to build ideal in vitro models in the future to help solve existing research challenges. This article briefly introduces the relevant 3D bioprinting technologies and bioinks that can be used by otorhinolaryngologists and discusses their potential applications in otorhinolaryngology.
... [39][40][41] Besides, microvalve-based bioprinting has a throughput of up to 1 kHz with a droplet diameter range of 100 to 600 μm. 39,[42][43][44] Limited by the droplet diameter, the tissues fabricated by microvalve-based bioprinting show a lower resolution than that by TIJ, PIJ, or EHDJ. ...
Cancer is now one of the leading causes of mortality worldwide, and the cancer treatment development is still slow due to the lack of efficient in vitro tumor models for studying tumorigenesis and facilitating drug development. Multicellular tumor spheroids can recapitulate the critical properties of tumors in vivo, including spatial organization, physiological responses, and metabolism, and are considered powerful platform for disease study and drug screening. Although several spheroid fabrication methods have been developed, most of them result in uncontrolled cell aggregations, yielding spheroids of variable size and function. Droplet-based bioprinting is capable of depositing cells in spatiotemporal manner so as to control the composition and distribution of printed biological constructs, thereby facilitating high-throughput fabrication of complicated and reproducible tumor spheroids. In this review, we introduce the progress of droplet-based bioprinting technology for the fabrication of tumor spheroids. First, different droplet-based bioprinting technologies are compared in terms of their strengths and shortcomings, which should be taken into account while fabricating tumor spheroids. Second, the latest advances in modeling distinct types of cancers and the enabled applications with tumor spheroids are summarized. Finally, we discuss the challenges and potentials revolving around the advances of bioprinting technology, improvement of spheroid quality, and integration of different technologies.
... The overarching aim of enhancing cell attachment, proliferation, and vascularization highlights the intricate balance of factors necessary for the success of tissue-engineered constructs [17]. The integration of 3D printing and biofabrication techniques has transformed the ability to create complex structures and precisely fabricate tissues [18][19][20]. Ongoing research into new materials and methodologies seeks to address the dynamic challenges faced in regenerative medicine [21,22]. ...
... Further research includes the 3D printing of cell-laden collagen hydrogel structures for regenerative medicine, which allows for uniform cell seeding and long-term viability [20]. The use of PDMS from laser-etched acrylic for making molds has enabled the control of tissue morphology in engineered cardiac tissues [59]. ...
... By encapsulating hESCs in dextran-based hydrogels enriched with regulatory factors, this method effectively increases the expression of vascular markers, demonstrating the potential for applications in vascular tissue engineering [69]. [20]. The use of PDMS from laser-etched acrylic for making molds has enabled the control of tissue morphology in engineered cardiac tissues [59]. ...
This manuscript covers the latest advancements and persisting challenges in the domain of tissue engineering, with a focus on the development and engineering of hydrogel scaffolds. It highlights the critical role of these scaffolds in emulating the native tissue environment, thereby providing a supportive matrix for cell growth, tissue integration, and reducing adverse reactions. Despite significant progress, this manuscript emphasizes the ongoing struggle to achieve an optimal balance between biocompatibility, biodegradability, and mechanical stability, crucial for clinical success. It also explores the integration of cutting-edge technologies like 3D bioprinting and biofabrication in constructing complex tissue structures, alongside innovative materials and techniques aimed at enhancing tissue growth and functionality. Through a detailed examination of these efforts, the manuscript sheds light on the potential of hydrogels in advancing regenerative medicine and the necessity for multidisciplinary collaboration to navigate the challenges ahead.
... The excess section is cut off by the MATLAB software when it determines the line segment between two intersecting planes. The stiffener is oriented at 15° degrees to the horizontal plane, and the object is cut horizontally and inclined (Moon et al., 2010). To show off the potential of multiple plane slicing and printing using an industrial robot, the stiffener is produced as a number of layers with gradations in size. ...
Due to the development of industrial 5.0 concepts, the integration of multi-disciplinary
concepts is evolving in the manufacturing industries. In this chapter, the integration
of robot technology with additive manufacturing processes is discussed to improve
the processes for making complex objects. A case study has been used to explain
two-phase and multi-plane three-dimensional printing using industrial robots.
Using previous research activities, a brief literature on 3D printing technology,
parameters, and applications has been developed. The case study on the aerofoil
wing made by industrial robotics programming has been explained. The flow chart
that can exhibit both three phase and multi-plane processes has been illustrated
... The development of various disease models is connected to interdisciplinary research activities such as mechanical, medical, electrical, material, tissue, and molecular engineers. Building the new concept and tissue models is important based on the damage to human organs (Moon et al., 2010). ...
The additive manufacturing technology has been applied in various sectors: the manufacturing of industrial components, toys, medicine, medical-surgical instruments, and tissue engineering sectors. In the tissue engineering field, it has been intensively applied to make biomaterials, organs, and drugs. The fundamental procedures of the additive manufacturing process, the various additive manufacturing techniques, and advanced methods that have been applied in the making and synthesis of organs in the tissue engineering fields have been described. In this chapter, the computer-aided tissue modelling process, different fundamental and advanced biomaterials, and advanced scaffold manufacturing applications in emerging tissue engineering fields have been illustrated.
... The widespread extrusionbased and droplet bioprinting technologies provide more homogenous, controllable size, cell number and shape distributions, as well as potentially "real" tissue formations for tumour biology studies. One of the first layer-by-layer cell printing applications described better cell seeding uniformity and longterm viability (>90%, 14 days) of the printed primary cells (62). Additionally, after 2010, in some studies, magnetic levitation of the tumour cells and fibroblasts was applied aiding the formation of tumour spheres (breast cancer) with defined cellular composition and density by Leonard's method (63). ...
Growing evidence propagates those alternative technologies (relevant human cell-based—e.g., organ-on-chips or biofabricated models—or artificial intelligence-combined technologies) that could help in vitro test and predict human response and toxicity in medical research more accurately. In vitro disease model developments have great efforts to create and serve the need of reducing and replacing animal experiments and establishing human cell-based in vitro test systems for research use, innovations, and drug tests. We need human cell-based test systems for disease models and experimental cancer research; therefore, in vitro three-dimensional (3D) models have a renaissance, and the rediscovery and development of these technologies are growing ever faster. This recent paper summarises the early history of cell biology/cellular pathology, cell-, tissue culturing, and cancer research models. In addition, we highlight the results of the increasing use of 3D model systems and the 3D bioprinted/biofabricated model developments. Moreover, we present our newly established 3D bioprinted luminal B type breast cancer model system, and the advantages of in vitro 3D models, especially the bioprinted ones. Based on our results and the reviewed developments of in vitro breast cancer models, the heterogeneity and the real in vivo situation of cancer tissues can be represented better by using 3D bioprinted, biofabricated models. However, standardising the 3D bioprinting methods is necessary for future applications in different high-throughput drug tests and patient-derived tumour models. Applying these standardised new models can lead to the point that cancer drug developments will be more successful, efficient, and consequently cost-effective in the near future.
... Applications of 3D bioprinting range from microfluidics, organ-on-chip technologies, and tissue engineering to real-sized organ implants [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21]. Commonly used 3D bioprinting methods are laser-assisted bioprinting [22], stereolithography (SLA) [17,23], inkjet-based bioprinting [24], valve-based bioprinting [25], and extrusion-based bioprinting (EBB) [26]. While laser-assisted and SLA (known as light-induced methods) possess high resolution and can fabricate complex 3D patterns, the high cost, limited material selection, scalability, and potential photo-induced cell damages are challenging. ...
Extrusion-based 3D bioprinting is a promising technique for fabricating multi-layered, complex biostructures, as it enables multi-material dispersion of bioinks with a straightforward procedure (particularly for users with limited additive manufacturing skills). Nonetheless, this method faces challenges in retaining the shape fidelity of the 3D-bioprinted structure, i.e., the collapse of filament (bioink) due to gravity and/or spreading of the bioink owing to the low viscosity, ultimately complicating the fabrication of multi-layered designs that can maintain the desired pore structure. While low viscosity is required to ensure a continuous flow of material (without clogging), a bioink should be viscous enough to retain its shape post-printing, highlighting the importance of bioink properties optimization. Here, two quantitative analyses are performed to evaluate shape fidelity. First, the filament collapse deformation is evaluated by printing different concentrations of alginate and its crosslinker (calcium chloride) by a co-axial nozzle over a platform to observe the overhanging deformation over time at two different ambient temperatures. In addition, a mathematical model is developed to estimate Young’s modulus and filament collapse over time. Second, the printability of alginate is improved by optimizing gelatin concentrations and analyzing the pore size area. In addition, the biocompatibility of proposed bioinks is evaluated with a cell viability test. The proposed bioink (3% w/v gelatin in 4% alginate) yielded a 98% normalized pore number (high shape fidelity) while maintaining >90% cell viability five days after being bioprinted. Integration of quantitative analysis/simulations and 3D printing facilitate the determination of the optimum composition and concentration of different elements of a bioink to prevent filament collapse or bioink spreading (post-printing), ultimately resulting in high shape fidelity (i.e., retaining the shape) and printing quality.
... The increasing demand for tissues or organs that meet the criteria to replace damaged or lost tissue or organ functions makes tissue engineering an encouraging technique to shape human organs and tissues [1][2][3]. Although the formation of the two-dimensional scaffold was successful in vitro through tissue engineering, it was inadequate to resemble the original tissue in a complex manner, nor did the perspective of three-dimensional (3D) polymer scaffolding [1,4]. ...
... The increasing demand for tissues or organs that meet the criteria to replace damaged or lost tissue or organ functions makes tissue engineering an encouraging technique to shape human organs and tissues [1][2][3]. Although the formation of the two-dimensional scaffold was successful in vitro through tissue engineering, it was inadequate to resemble the original tissue in a complex manner, nor did the perspective of three-dimensional (3D) polymer scaffolding [1,4]. Therefore, the 3D bioprinting approach has been developed to overcome various shortcomings of tissue engineering, especially in the formation of a stable scaffold with biocompatibility for cell survival, which allows the fabrication of multicellular tissues needed in copious tissue engineering applications [1,[5][6][7][8][9][10][11]. ...
... Although the formation of the two-dimensional scaffold was successful in vitro through tissue engineering, it was inadequate to resemble the original tissue in a complex manner, nor did the perspective of three-dimensional (3D) polymer scaffolding [1,4]. Therefore, the 3D bioprinting approach has been developed to overcome various shortcomings of tissue engineering, especially in the formation of a stable scaffold with biocompatibility for cell survival, which allows the fabrication of multicellular tissues needed in copious tissue engineering applications [1,[5][6][7][8][9][10][11]. Forming organs or tissues that are following the complex microarchitecture of native tissue through a bioprinting approach face various challenges in overcoming the low level of biocompatibility to cells, which leads to a loss or damage to cell function, as well as blockages during the printing process [1,2,[12][13][14]. ...
The present study was to investigate the rheological property, printability, and cell viability of alginate–gelatin composed hydrogels as a potential cell-laden bioink for three-dimensional (3D) bioprinting applications. The 2 g of sodium alginate dissolved in 50 mL of phosphate buffered saline solution was mixed with different concentrations (1% (0.5 g), 2% (1 g), 3% (1.5 g), and 4% (2 g)) of gelatin, denoted as GBH-1, GBH-2, GBH-3, and GBH-4, respectively. The properties of the investigated hydrogels were characterized by contact angle goniometer, rheometer, and bioprinter. In addition, the hydrogel with a proper concentration was adopted as a cell-laden bioink to conduct cell viability testing (before and after bioprinting) using Live/Dead assay and immunofluorescence staining with a human corneal fibroblast cell line. The analytical results indicated that the GBH-2 hydrogel exhibited the lowest loss rate of contact angle (28%) and similar rheological performance as compared with other investigated hydrogels and the control group. Printability results also showed that the average wire diameter of the GBH-2 bioink (0.84 ± 0.02 mm (*** p < 0.001)) post-printing was similar to that of the control group (0.79 ± 0.05 mm). Moreover, a cell scaffold could be fabricated from the GBH-2 bioink and retained its shape integrity for 24 h post-printing. For bioprinting evaluation, it demonstrated that the GBH-2 bioink possessed well viability (>70%) of the human corneal fibroblast cell after seven days of printing under an ideal printing parameter combination (0.4 mm of inner diameter needle, 0.8 bar of printing pressure, and 25 °C of printing temperature). Therefore, the present study suggests that the GBH-2 hydrogel could be developed as a potential cell-laden bioink to print a cell scaffold with biocompatibility and structural integrity for soft tissues such as skin, cornea, nerve, and blood vessel regeneration applications.
... This technology, based on living cell cultures, biocompatible materials and digital support tools, enables the layer-by-layer arrangement of biomaterials, biochemicals and living cells with accurate spatial control, thus mimicking the systemic complexities of conditions physiological or pathological (Guillotin et al., 2010;Moon et al., 2010). ...
Recently, research is undergoing a drastic change in the application of the animal model as a unique investigation strategy, considering an alternative approach for the development of science for the future. Although conventional monolayer cell cultures represent an established and widely used in vitro method, the lack of tissue architecture and the complexity of such a model fails to inform true biological processes in vivo. Recent advances in cell culture techniques have revolutionized in vitro culture tools for biomedical research by creating powerful three-dimensional (3D) models to recapitulate cell heterogeneity, structure and functions of primary tissues. These models also bridge the gap between traditional two-dimensional (2D) single-layer cultures and animal models. 3D culture systems allow researchers to recreate human organs and diseases in one dish and thus holds great promise for many applications such as regenerative medicine, drug discovery, precision medicine, and cancer research, and gene expression studies. Bioengineering has made an important contribution in the context of 3D systems using scaffolds that help mimic the microenvironments in which cells naturally reside, supporting the mechanical, physical and biochemical requirements for cellular growth and function. We therefore speak of models based on organoids, bioreactors, organ-on-a-chip up to bioprinting and each of these systems provides its own advantages and applications. All of these techniques prove to be excellent candidates for the development of alternative methods for animal testing, as well as revolutionizing cell culture technology. 3D systems will therefore be able to provide new ideas for the study of cellular interactions both in basic and more specialized research, in compliance with the 3R principle. In this review, we provide a comparison of 2D cell culture with 3D cell culture, provide details of some of the different 3D culture techniques currently available by discussing their strengths as well as their potential applications.
... Bioprinting technology has attracted extensive attention in recent years. Bioprinting is defined as the use of 3D printing technology to deposit bioink or biomaterials ink on a receiving solid or gel substrate or liquid reservoir (Demirci and Montesano, 2007;Mironov et al., 2008;Moon et al., 2010).3D printing technology is an emerging technology, which can accurately "reproduce" tissue using the computer-aided design (CAD). ...
Spinal cord injury (SCI) is considered to be one of the most challenging central nervous system injuries. The poor regeneration of nerve cells and the formation of scar tissue after injury make it difficult to recover the function of the nervous system. With the development of tissue engineering, three-dimensional (3D) bioprinting has attracted extensive attention because it can accurately print complex structures. At the same time, the technology of blending and printing cells and related cytokines has gradually been matured. Using this technology, complex biological scaffolds with accurate cell localization can be manufactured. Therefore, this technology has a certain potential in the repair of the nervous system, especially the spinal cord. So far, this review focuses on the progress of tissue engineering of the spinal cord, landmark 3D bioprinting methods, and landmark 3D bioprinting applications of the spinal cord in recent years.
