(In progress)

Table of Contents

Special Section: Bioprinting of 3D Functional Tissue Constructs

Regular Section

Review article

by Xiaorui Li, Fuyin Zheng, Xudong Wang, Xuezheng Geng, Shudong Zhao, Hui Liu, Dandan Dou, Yubing Leng, Lizhen Wang, Yubo Fan

Three-dimensional (3D) extrusion-based bioprinting is the most widely used bioprinting technology to fabricate bionic tissue or organ constructs by combining biomaterial ink and living cells for tissue engineering and regenerative medicine. One critical issue of this technique is the selection of suitable biomaterial ink to simulate extracellular matrix (ECM) that provides mechanical support for cells and regulates their physiological activities. Previous studies have demonstrated that it is an enormous challenge to form and maintain reproducible 3D constructs and eventually achieve the balance among biocompatibility, mechanical properties, and printability. This review highlights the properties of extrusion-based biomaterial inks and recent developments as well as details various biomaterial inks classified by their function. Key approaches related to their modification methods according to the functional requirements are also discussed, along with the selection strategies by varying extrusion paths and methods in extrusion-based bioprinting. This systematical review will assist researchers in identifying the most suitable extrusion based biomaterial inks based on their requirements, as well as in elaborating current challenges and prospects of extrudable biomaterial inks in the field of bioprinting of in vitro tissue models.

Research article

by Alejandro Madrid-Sánchez, Fabian Duerr, Fabian Duerr, Yunfeng Nie, Yunfeng Nie, Hugo Thienpont, Hugo Thienpont, Heidi Ottevaere, Heidi Ottevaere

The common characteristics that make scaffolds suitable for human tissue substitutes include high porosity, microscale features, and pores interconnectivity. Too often, however, these characteristics are limiting factors for the scalability of different fabrication approaches, particularly in bioprinting techniques, in which either poor resolution, small areas, or slow processes hinder practical use in certain applications. An excellent example is bioengineered scaffolds for wound dressings, in which microscale pores in large surface-to-volume ratio scaffolds must be manufactured – ideally fast, precise, and cheap, and where conventional printing methods do not readily meet both ends. In this work, we propose an alternative vat photopolymerization technique to fabricate centimeter-scale scaffolds without losing resolution. We used laser beam shaping to first modify the profile of the voxels in 3D printing, resulting in a technology we refer to as light sheet stereolithography (LS-SLA). For proof of concept, we developed a system from commercially available off-the-shelf components to demonstrate strut thicknesses up to 12.8 ± 1.8 μm, tunable pore sizes ranging from 36 μm to 150 μm, and scaffold areas up to 21.4 mm × 20.6 mm printed in a short time. Furthermore, the potential to fabricate more complex and three-dimensional scaffolds was demonstrated with a structure composed of six layers, each rotated by 45° with respect to the previous. Besides the demonstrated high resolution and achievable large scaffold sizes, we found that LS-SLA has great potential for scaling-up of applied oriented technology for tissue engineering applications.

 

Research article

by Su Hee Kim, Se Jun Park, Bin Xu, Jae Hyup Lee, Sang Jin An, Misun Cha

Three-dimensional (3D) bioprinter including screw extruder was developed, and the polycaprolactone (PCL) grafts fabricated by screw-type and pneumatic pressure-type bioprinters were comparatively evaluated. The density and tensile strength of the single layers printed by the screw-type were 14.07% and 34.76% higher, respectively, than those of the single layers produced by the pneumatic pressure-type. The adhesive force, tensile strength, and bending strength of the PCL grafts printed by the screw-type bioprinter were 2.72 times, 29.89%, and 67.76% higher, respectively, than those of the PCL grafts prepared by the pneumatic pressure-type bioprinter. By evaluating the consistency with the original image of the PCL grafts, we found that it had a value of about 98.35%. The layer width of the printing structure was 485.2 ± 0.004919 μm, which was 99.5% to 101.8% compared to the set value (500 μm), indicating high accuracy and uniformity. The printed graft had no cytotoxicity, and there were no impurities in the extract test. In the in vivo studies, the tensile strength of the sample 12 months after implantation was reduced by 50.37% and 85.43% compared to the initial point of the sample printed by the screw-type and the pneumatic pressure-type, respectively. Through observing the fractures of the samples at 9- and 12-month samples, we found that the PCL grafts prepared by the screw-type had better in vivo stability. Therefore, the printing system developed in this study can be used as a treatment for regenerative medicine.

Review article

by Shuduan Mao, Junjie Man, Jialei Wang, Li Fu, Chengliang Yin, Hassan Karimi-Maleh

As the body’s largest organ, the skin has important roles in barrier function, immune response, prevention of water loss and excretion of waste. Patients with extensive and severe skin lesions would die due to insufficient graftable skin. Commonly used treatments include autologous skin grafts, allogeneic/allogeneic skin grafts, cytoactive factors, cell therapy, and dermal substitutes. However, traditional treatment methods are still inadequate regarding skin repair time, treatment costs, and treatment results. In recent years, the rapid development of bioprinting technology has provided new ideas to solve the above-mentioned challenges. This review describes the principles of bioprinting technology and research advances in wound dressing and healing. This review features a data mining and statistical analysis of this topic through bibliometrics. The annual publications on this topic, participating countries, and institutions were used to understand the development history. Keyword analysis was used to understand the focus of investigation and challenges in this topic. According to bibliometric analysis, bioprinting in wound dressing and healing is in an explosive phase, and future research should focus on discovering new cell sources, innovative bioink development, and developing largescale printing technology processes.

Clinical case study

by Yongqiang Hao, Bojun Cao, Liang Deng, Jiaxin Li, Zhaoyang Ran, Junxiang Wu, Boran Pang, Jia Tan, Dinghao Luo, Wen Wu
The repair and reconstruction of bone defects are still major problems to be solved in the field of orthopedics. Meanwhile, 3D-bioprinted active bone implants may provide a new and effective solution. In this case, we used bioink prepared from the patient’s autologous platelet-rich plasma (PRP) combined with polycaprolactone/β-tricalcium phosphate (PCL/β-TCP) composite scaffold material to print personalized PCL/β-TCP/PRP active scaffolds layer by layer through 3D bioprinting technology. The scaffold was then applied in the patient to repair and reconstruct bone defect after tibial tumor resection. Compared with traditional bone implant materials, 3D-bioprinted personalized active bone will have significant clinical application prospects due to its advantages of biological activity, osteoinductivity, and personalized design.

Research article

by Matthias Gielisch, Diana Heimes, Daniel G. E. Thiem, C. Boesing, M. Krumpholtz, B. Al-Nawas, Peer W. Kämmerer

Three-dimensional (3D) printing is a rapidly evolving field and has gained increasing importance in the medical sector. However, the increasing usage of printing materials is accompanied by more wastages. With a rising awareness of the environmental impact of the medical sector, the development of highly accurate and biodegradable materials is of great interest. This study aims to compare the accuracy of polylactide/polyhydroxyalkanoate (PLA/PHA) surgical guides printed by fused filament fabrication and material jetted guides of MED610 in fully guided dental implant placement before and after steam sterilization. Five guides were tested in this study and each was either printed with PLA/PHA or MED610 and either steam-sterilized or not. After implant insertion in a 3D-printed upper jaw model, the divergence between planned and achieved implant position was calculated by digital superimposition. Angular deviation and 3D deviation at the base and the apex were determined. Non-sterilized PLA/PHA guides showed an angle deviation of 0.38 ± 0.53° compared to 2.88 ± 0.75° in sterile guides (P > 0.001), an offset of 0.49 ± 0.21 mm and 0.94 ± 0.23 mm (P < 0.05), and an offset at the apex of 0.50 ± 0.23 mm before and 1.04 ± 0.19 mm after steam sterilization (P < 0.025). No statistically significant difference could be shown for angle deviation or 3D offset at both locations for guides printed with MED610. PLA/PHA printing material showed significant deviations in angle and 3D accuracy after sterilization. However, the reached accuracy level is comparable to levels reached with materials already used in clinical routine and therefore, PLA/PHA surgical guide is a convenient and green alternative.

Research article

by Kai Cao, Fucheng Zhang, Ahmadreza Zaeri, Ralf Zgeib, Robert C. Chang

The printing accuracy of polymer melt electrowriting is adversely affected by the residual charge entrapped within the fibers, especially for three-dimensional (3D) structured materials or multilayered scaffolds with small interfiber distances. To clarify this effect, an analytical charge-based model is proposed herein. The electric potential energy of the jet segment is calculated considering the amount and distribution of the residual charge in the jet segment and the deposited fibers. As the jet deposition proceeds, the energy surface assumes different patterns, which constitute different modes of evolution. The manner in which the various identified parameters affect the mode of evolution are represented by three charge effects, including the global, local, and polarization effect. Based on these representations, typical modes of energy surface evolution are identified. Moreover, the lateral characteristic curve and characteristic surface are advanced to analyze the complex interplay between fiber morphologies and residual charge. Different parameters contribute to this interplay either by affecting residual charge, fiber morphologies, or the three charge effects. To validate this model, the effects of lateral location and grid number (i.e., number of fibers printed in each direction) on the fiber morphologies are investigated. Moreover, the “fiber bridging” phenomenon in parallel fiber printing is successfully explained. These results help to comprehensively understand the complex interplay between the fiber morphologies and the residual charge, thus furnishing a systematic workflow to improve printing accuracy.

Research article

by Zhaoxuan Feng, Jinsong Li, Dasen Zhou, Hui Song, Jiaqi Lv, Wenqin Bai

Digital light processing has significant advantages, such as high repeatability, low failure rate, and no extrusion shear force. To make it possible to print complex structures with high resolution, the consecutive development of photocurable ink materials is indispensable. In this work, photo-functionalized pullulan (Pul-NB) was prepared by introducing norbornene groups into pullulan chains, and an ink material suitable for photocurable printing was prepared by thiol-ene click reaction. The rheology, water absorption, and mechanical properties of the Pul-NB precursor solution and photocurable hydrogel were investigated. The optimal composition of Pul-NB ink for three-dimensional (3D) printing was obtained by adjusting the degree of substitution, Pul-NB concentration, and thiol crosslinking agent. This novel bioink for digital light processing 3D printing showed good printability and high shape fidelity. This ink material provides an excellent alternative for printing biomimetic soft tissue organs, high-throughput tissue models, soft robots, etc.

Research article

by Guoqing Zhang, Junxin Li, Xiaoyu Zhou, Yongsheng Zhou, Yuchao Bai
In order to generate a high-performance personalized biological fixation plate with matching mechanical properties and biocompatibility, reverse reconstruction and fracture reduction of a femur were performed by combining reverse and forward approaches, and the surface was extracted according to the installation position of the plate to complete plate modeling by shifting, thickening, and performing other operations. Subsequently, topology optimization and three-dimensional (3D) print¬ing were performed, and the properties of the manufactured plate were probed. The results showed that the maximum displacement of the plate was 4.13 mm near the femoral head, the maximum stress was 5.15e2 MPa on both sides of the plate across its entire length, and the stress concentration decreased following topology opti¬mization. The plate with optimized topology and filled with porous structure has a good filling effect. The final mass of the H-shaped plate was 12.05 g, while that of the B-shaped plate was 11.05 g, which dropped by 20.93% and 27.49%, respective¬ly, compared with the original plate. The surface of the 3D-printed plate was bright and new, with a clear pore structure and good lap joint. The B-shaped and H-shaped plates were closely dovetailed with the host bone, which met the assembly require¬ments. This lays a foundation for the direct application of a high-performance per¬sonalized biological fixation plate.

Research article

by Xingliang Dai, Xuefeng Tian, Shengcai Gu, Yafei Yang, Huaixu Li, Peng Gao, Hongwei Cheng, Qing Lan

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

 

Research article

by Jueun Kim, Yeong-Jin Choi, Chang-Woo Gal, Aram Sung, Honghyun Park, Hui-Suk Yun

Hydrogels are natural bioink options for cellular printing due to their high-water content and permeable three-dimensional (3D) polymeric structure, which are favorable for cellular anchoring and metabolic activities. To increase the functionality of hydrogels as bioinks, biomimetic components are often incorporated, such as proteins, peptides, and growth factors. In this study, we aimed to enhance the osteogenic activity of a hydrogel formulation by integrating both the release and retention of gelatin so that gelatin serves as both an indirect support for released ink component on cells nearby and a direct support for encapsulated cells inside a printed hydrogel, thereby fulfills two functions. Methacrylate-modified alginate (MA-alginate) was chosen as the matrix because it has a low cell adhesion effect due to the absence of ligands. The gelatin-containing MA-alginate hydrogel was fabricated, and gelatin was found to remain in the hydrogel for up to 21 days. The gelatin remaining in the hydrogel had positive effects on encapsulated cells, especially on cell proliferation and osteogenic differentiation. The gelatin released from the hydrogel affected the external cells, showing more favorable osteogenic behavior than the control sample. It was also found that the MA-alginate/gelatin hydrogel could be used as a bioink for printing with high cell viability. Therefore, we anticipate that the alginate-based bioink developed in this study could potentially be used to induce osteogenesis in bone tissue regeneration.

Research article

by Eben Adarkwa, Abhijit Roy, John Ohodnicki, Boeun Lee, Prashant N. Kumta, Salil Desai

Three-dimensional (3D) printing is implemented for surface modification of titanium alloy substrates with multilayered biofunctional polymeric coatings. Poly(lactic-co-glycolic) acid (PLGA) and polycaprolactone (PCL) polymers were embedded with amorphous calcium phosphate (ACP) and vancomycin (VA) therapeutic agents to promote osseointegration and antibacterial activity, respectively. PCL coatings revealed a uniform deposition pattern of the ACP-laden formulation and enhanced cell adhesion on the titanium alloy substrates as compared to the PLGA coatings. Scanning electron microscopy and Fourier-transform infrared spectroscopy confirmed a nanocomposite structure of ACP particles showing strong binding with the polymers. Cell viability data showed comparable MC3T3 osteoblast proliferation on polymeric coatings as equivalent to positive controls. In vitro live/dead assessment indicated higher cell attachments for 10 layers (burst release of ACP) as compared to 20 layers (steady release) for PCL coatings. The PCL coatings loaded with the antibacterial drug VA displayed a tunable release kinetics profile based on the multilayered design and drug content of the coatings. Moreover, the concentration of active VA released from the coatings was above the minimum inhibitory concentration and minimum bactericidal concentration, demonstrating its effectiveness against Staphylococcus aureus bacterial strain. This research provides a basis for developing antibacterial biocompatible coatings to promote osseointegration of orthopedic implants

Research article

by Zijie Pei, Mingyang Gao, Junhui Xing, Changbao Wang, Piqian Zhao, Hongtao Zhang, Jing Qu

Cartilage damage is a common orthopedic disease, which can be caused by sports injury, obesity, joint wear, and aging, and cannot be repaired by itself. Surgical autologous osteochondral grafting is often required in deep osteochondral lesions to avoid the later progression of osteoarthritis. In this study, we fabricated a gelatin methacryloyl-marrow mesenchymal stem cells (GelMA-MSCs) scaffold by three-dimensional (3D) bioprinting. This bioink is capable of fast gel photocuring and spontaneous covalent cross-linking, which can maintain high viability of MSCs and provide a benign microenvironment to promote the interaction, migration, and proliferation of cells. In vivo experiments, further, proved that the 3D bioprinting scaffold can promote the regeneration of cartilage collagen fibers and have a remarkable effect on cartilage repair of rabbit cartilage injury model, which may represent a general and versatile strategy for precise engineering of cartilage regeneration system.

Review article

by Diána Szűcs, Zsolt Fekete, Melinda Guba, Lajos Kemény, Katalin Jemnitz, Emese Kis, Zoltán Veréb
The importance of three-dimensional (3D) models in pharmacological tests and personalized therapies is significant. These models allow us to gain insight into the cell response during drug absorption, distribution, metabolism, and elimination in an organ-like system and are suitable for toxicological testing. In personalized and regenerative medicine, the precise characterization of artificial tissues or drug metabolism processes is more than crucial to gain the safest and the most effective treatment for the patients. Using these 3D cell cultures derived directly from patient, such as spheroids, organoids, and bioprinted structures, allows for testing drugs before administration to the patient. These methods allow us to select the most appropriate drug for the patient. Moreover, they provide chance for better recovery of patients, since time is not wasted during therapy switching. These models could be used in applied and basic research as well, because their response to treatments is quite similar to that of the native tissue. Furthermore, they may replace animal models in the future because these methods are cheaper and can avoid interspecies differences. This review puts a spotlight on this dynamically evolving area and its application in toxicological testing.

Review article

by Fatima Garcia-Villen, Fernando López-Zárraga, Cesar Viseras, Sandra Ruiz-Alonso, Fouad Al-Hakim, Irene Diez-Aldama, Laura Saenz-del-Burgo, Denis Scaini, Jose Luis Pedraz

Vascular stents (VS) have revolutionized the treatment of cardiovascular diseases, as evidenced by the fact that the implantation of VS in coronary artery disease (CAD) patients has become a routine, easily approachable surgical intervention for the treatment of stenosed blood vessels. Despite the evolution of VS throughout the years, more efficient approaches are still required to address the medical and scientific challenges, especially when it comes to peripheral artery disease (PAD). In this regard, three-dimensional (3D) printing is envisaged as a promising alternative to upgrade VS by optimizing the shape, dimensions and stent backbone (crucial for optimal mechanical properties), making them customizable for each patient and each stenosed lesion. Moreover, the combination of 3D printing with other methods could also upgrade the final device. This review focuses on the most recent studies using 3D printing techniques to produce VS, both by itself and in combination with other techniques. The final aim is to provide an overview of the possibilities and limitations of 3D printing in the manufacturing of VS. Furthermore, the current situation of CAD and PAD pathologies is also addressed, thus highlighting the main weaknesses of the already existing VS and identifying research gaps, possible market niches and future directions.

Review article

by Shahram Parvaneh, Lajos Kemény, Ameneh Ghaffarinia, Reza Yarani, Zoltán Veréb

Diabetes is an autoimmune disease that ensues when the pancreas does not deliver adequate insulin or when the body cannot react to the existing insulin. Type 1 diabetes is an autoimmune disease defined by continuous high blood sugar levels and insulin deficiency due to β-cell destruction in the islets of Langerhans (pancreatic islets). Long-term complications, such as vascular degeneration, blindness, and renal failure, result from periodic glucose-level fluctuations following exogenous insulin therapy. Nevertheless, the shortage of organ donors and the lifelong dependency on immunosuppressive drugs limit the transplantation of the entire pancreas or pancreas islet, which is the therapy for this disease. Although encapsulating pancreatic islets using multiple hydrogels creates a semi-privileged environment to prevent immune rejection, hypoxia that occurs in the core of the capsules is the main hindrance that should be solved. Bioprinting technology is an innovative process in advanced tissue engineering that allows the arranging of a wide array of cell types, biomaterials, and bioactive factors as a bioink to simulate the native tissue environment for fabricating clinically applicable bioartificial pancreatic islet tissue. Multipotent stem cells have the potential to be a possible solution for donor scarcity and can be a reliable source for generating autograft and allograft functional β-cells or even pancreatic islet-like tissue. The use of supporting cells, such as endothelial cells, regulatory T cells, and mesenchymal stem cells, in the bioprinting of pancreatic islet-like construct could enhance vasculogenesis and regulate immune activity. Moreover, scaffolds bioprinted using biomaterials that can release oxygen postprinting or enhance angiogenesis could increase the function of β-cells and the survival of pancreatic islets, which could represent a promising avenue.

Research article

by Jesús M. Rodríguez-Rego, Laura Mendoza-Cerezo, Antonio Macías-García, Juan P. Carrasco-Amador, Alfonso C. Marcos-Romero

Currently, the characterization techniques for hydrogels used in bioprinting are extensive, and they could provide data on the physical, chemical, and mechanical properties of hydrogels. While characterizing the hydrogels, the analysis of their printing properties is of great importance in the determination of their potential for bioprinting. The study of printing properties provides data on their capacity to reproduce biomimetic structures and maintain their integrity after the process, as it also relates them to the possible cell viability after the generation of the structures. Current hydrogel characterization techniques require expensive measuring instrument that is not readily available in many research groups. Therefore, it would be interesting to propose a methodology to characterize and compare the printability of different hydrogels in a fast, simple, reliable, and inexpensive way. The aim of this work is to propose a methodology for extrusion-based bioprinters that allows determining the printability of hydrogels that are going to be loaded with cells, by analyzing cell viability with the sessile drop method, molecular cohesion with the filament collapse test, adequate gelation with the quantitative evaluation of the gelation state, and printing precision with the printing grid test. The data obtained after performing this work allow the comparison of different hydrogels or different concentrations of the same hydrogel to determine which one has the most favorable properties to carry out bioprinting studies.

Research article

by Zhijing He, Chen Jiao, Junnan Wu, Jiasen Gu, Huixin Liang, Lida Shen, Youwen Yang, Zongjun Tian, Changjiang Wang, Qing Jiang
Porous hydroxyapatite (HA) scaffolds prepared by three-dimensional (3D) printing have wide application prospects owing to personalized structural design and excellent biocompatibility. However, the lack of antimicrobial properties limits its widespread use. In this study, a porous ceramic scaffold was fabricated by digital light processing (DLP) method. The multilayer chitosan/alginate composite coatings prepared by layer-by-layer method were applied to scaffolds and Zn2+ was doped into coatings in the form of ion crosslinking. The chemical composition and morphology of coatings were characterized by scanning electron microscope (SEM) and X-ray photoelectron spectroscopy (XPS). Energy dispersive spectroscopy (EDS) analysis demonstrated that Zn2+ was uniformly distributed in the coating. Besides, the compressive strength of coated scaffolds (11.52 ± 0.3 MPa) was slightly improved compared with that of bare scaffolds (10.42 ± 0.56 MPa). The result of soaking experiment indicated that coated scaffolds exhibited delayed degradation. In vitro experiments demonstrated that within the limits of concentration, a higher Zn content in the coating has a stronger capacity to promote cell adhesion, proliferation and differentiation. Although excessive release of Zn2+ led to cytotoxicity, it presented a stronger antibacterial effect against Escherichia coli (99.4%) and Staphylococcus aureus (93%).

Research article

by Reinhard Kaufmann, Michael Deutschmann, Matthias Meissnitzer, Bernhard Scharinger, Klaus Hergan, Andreas Vötsch, Christian Dinges, Stefan Hecht

Three-dimensional (3D)-printed vascular models for cardiovascular surgery planning and endovascular procedure simulations often lack realistic biological tissues mim¬icking material properties, including flexibility or transparency, or both. Transparent silicone or silicone-like vascular models were not available for end-user 3D printers and had to be fabricated using complex and cost-intensive workarounds. This limita¬tion has now been overcome by novel liquid resins with biological tissue properties. These new materials enable simple and low-cost fabrication of transparent and flexi¬ble vascular models using end-user stereolithography 3D printers and are promising technological advances toward more realistic patient-specific, radiation-free proce¬dure simulations and planning in cardiovascular surgery and interventional radiol¬ogy. This paper presents our patient-specific manufacturing process of fabricating transparent and flexible vascular models using freely available open-source software for segmentation and 3D post-processing, aiming to facilitate the integration of 3D printing into clinical care.

Research article

by Assaf Bar, Olga Kryukov, Sharon Etzion, Smadar Cohen

 In recent years, extrusion-based three-dimensional (3D) bioprinting is employed for engineering cardiac patches (CP) due to its ability to assemble complex structures from hydrogel-based bioinks. However, the cell viability in such CPs is low due to shear forces applied on the cells in the bioink, inducing cellular apoptosis. Herein, we investigated whether the incorporation of extracellular vesicles (EVs) in the bioink, engineered to continually deliver the cell survival factor miR-199a-3p would increase the viability within the CP. EVs from THP-1-derived activated macrophages (MΦ) were isolated and characterized by nanoparticle tracking analysis (NTA), cryogenic electron microscopy (cryo-TEM), and Western blot analysis. MiR-199a-3p mimic was loaded into EVs by electroporation after optimization of applied voltage and pulses. Functionality of the engineered EVs was assessed in neonatal rat cardiomyocyte (NRCM) monolayers using immunostaining for the proliferation markers ki67 and Aurora B kinase. To examine the effect of engineered EVs on 3D-bioprinted CP viability, the EVs were added to the bioink, consisting of alginate-RGD, gelatin, and NRCM. Metabolic activity and expression levels of activated-caspase 3 for apoptosis of the 3D-bioprinted CP were evaluated after 5 days. Electroporation (850 V with 5 pulses) was found to be optimal for miR loading; miR-199a-3p levels in EVs increased fivefold compared to simple incubation, with a loading efficiency of 21.0%. EV size and integrity were maintained under these conditions. Cellular uptake of engineered EVs by NRCM was validated, as 58% of cTnT+ cells internalized EVs after 24 h. The engineered EVs induced CM proliferation, increasing the ratio of cell-cycle re-entry of cTnT+ cells by 30% (Ki67) and midbodies+ cell ratio by twofold (Aurora B) compared with the controls. The inclusion of engineered EVs in bioink yielded CP with threefold greater cell viability compared to bioink with no EVs. The prolonged effect of EVs was evident as the CP exhibited elevated metabolic activities after 5 days, with less apoptotic cells compared to CP with no EVs. The addition of miR-199a-3p–loaded EVs to the bioink improved the viability of 3D-printed CP and is expected to contribute to their integration in vivo.

Research article

by Yunxia Liang, Bimal Chitrakar, Zhenbin Liu, Xujia Ming, Dan Xu, Haizhen Mo, Chunyang Shi, Xiaolin Zhu, Liangbin Hu, Hongbo Li
Benzyl isothiocyanate (BITC) is an isothiocyanate of plant origin, especially the mustard family, which has good antibacterial properties. However, its applications are challenging due to its poor water solubility and chemical instability. We used food hydrocolloids, including xanthan gum, locust bean gum, konjac glucomannan, and carrageenan as three-dimensional (3D)-printing food ink base and successfully prepared 3D-printed BITC antibacterial hydrogel (BITC-XLKC-Gel). The characterization and fabrication procedure of BITC-XLKC-Gel was studied. The results show that BITC-XLKC-Gel hydrogel has better mechanical properties by low-field nuclear magnetic resonance (LF-NMR), mechanical properties, and rheometer analysis. The strain rate of BITC-XLKC-Gel hydrogel is 76.5%, which is better than that of human skin. Scanning electron microscope (SEM) analysis showed that BITC-XLKC-Gel has uniform pore size and provides a good carrier environment for BITC carriers. In addition, BITC-XLKC-Gel has good 3D-printing performance, and 3D printing can be used for customizing patterns. Finally, inhibition zone analysis showed that the BITC-XLKC-Gel added with 0.6% BITC had strong antibacterial activity against Staphylococcus aureus and the BITC-XLKC-Gel added with 0.4% BITC had strong antibacterial activity against Escherichia coli. Antibacterial wound dressing has always been considered essential in burn wound healing. In experiments that simulated burn infection, BITC-XLKC-Gel showed good antimicrobial activity against methicillin-resistant S. aureus. BITC-XLKC-Gel is a good 3D-printing food ink attributed to strong plasticity, high safety profile, and good antibacterial performance and has great application prospects.

Research article

by Lothar Koch, Andrea Deiwick, Jordi Soriano, Boris Chichkov


Generation of human neuronal networks by three-dimensional (3D) bioprinting is promising for drug testing and hopefully will allow for the understanding of cellular mechanisms in brain tissue. The application of neural cells derived from human induced-pluripotent stem cells (hiPSCs) is an obvious choice, since hiPSCs provide access to cells unlimited in number and cell types that could be generated by differentiation. The questions in this regard include which neuronal differentiation stage is optimal for printing of such networks, and to what extent the addition of other cell types, especially astrocytes, supports network formation. These aspects are the focus of the present study, in which we applied a laser-based bioprinting technique and compared hiPSC-derived neural stem cells (NSCs) with neuronal differentiated NSCs, with and without the inclusion of co-printed astrocytes. In this study, we investigated in detail the effects of cell types, printed droplet size, and duration of differentiation before and after printing on viability, as well as proliferation, stemness, differentiation potential, formation of dendritic extensions and synapses, and functionality of the generated neuronal networks. We found a significant dependence of cell viability after dissociation on differentiation stage, but no impact of the printing process. Moreover, we observed a dependence of the abundance of neuronal dendrites on droplet size, a marked difference between printed cells and normal cell culture in terms of further differentiation of the cells, especially differentiation into astrocytes, as well as neuronal network formation and activity. Notably, there was a clear effect of admixed astrocytes on NSCs but not on neurons.

Research article

by Xulin Hu, Hu Li, Liang Qiao, Shuhao Yang, Haoming Wu, Chao Peng, Yamei Zhang, Hai Lan, Hua Yang, Kainan Li

 Human bone is composed of cortical bone and cancellous bone. The interior portion of natural bone is cancellous with a porosity of 50%–90%, but the outer layer is made of dense cortical bone, of which porosity was not higher than 10%. Porous ceramics were expected to be research hotspot in bone tissue engineering by virtue of their similarity to the mineral constituent and physiological structure of human bone. However, it is challenging to utilize conventional manufacturing methods to fabricate porous structures with precise shapes and pore sizes. Three-dimensional (3D) printing of ceramics is currently the latest research trend because it has many advantages in the fabrication of porous scaffolds, which can meet the requirements of cancellous bone strength, arbitrarily complex shapes, and individualized design. In this study, β-tricalcium phosphate (β-TCP)/titanium dioxide (TiO2) porous ceramics scaffolds were fabricated by 3D gel-printing sintering for the first time. The chemical constituent, microstructure, and mechanical properties of the 3D-printed scaffolds were characterized. After sintering, a uniform porous structure with appropriate porosity and pore sizes was observed. Besides, biological mineralization activity and biocompatibility were evaluated by in vitro cell assay. The results demonstrated that the incorporation of TiO2 (5 wt%) significantly improved the compressive strength of the scaffolds, with an increase of 283%. Additionally, the in vitro results showed that the β-TCP/TiO2 scaffold had no toxicity. Meanwhile, the adhesion and proliferation of MC3T3-E1 cells on scaffolds were desirable, revealing that the β-TCP/TiO2 scaffolds can be used as a promising candidate for repair scaffolding in orthopedics and traumatology.