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Special Section: Bioprinting of 3D Functional Tissue Constructs

Regular Section

Original research article

by Shugang Hu, Zijie Meng, Junpeng Zhou, Yongwei Li, Yanwen Su, Qi Lei, Mao Mao, Xiaoli Qu, Jiankang He, Wei Wang

Micro/sub-microscale fibrillar architectures of extracellular matrix play important roles in regulating cellular behaviors such as attachment, migration, and differentiation. However, the interactions between cells and organized micro/sub-microscale fibers have not been fully clarified yet. Here, the responses of MC3T3-E1 cells to electrohydrodynamic (EHD) printed scaffolds with microscale and/or sub-microscale fibrillar architectures were investigated to demonstrate their potential for bone tissue regeneration. Fibrillar scaffolds were EHD-fabricated with microscale (20.51 ± 1.70 μm) and/or sub-microscale (0.58 ± 0.51 μm) fibers in a controlled manner. The in vitro results showed that cells exhibited a 1.25-fold increase in initial attached cell number and 1.17-fold increase in vinculin expression on scaffolds with micro/sub-microscale fibers than that on scaffolds with pure microscale fibers. After 14 days of culture, the cells expressed 1.23 folds increase in collagen type I (COL-I) deposition compared with that on scaffolds with pure microscale fibers. These findings indicated that the EHD printed sub-microscale fibrous architectures can facilitate attachment and COL I secretion of MC3T3-E1 cells, which may provide a new insight to the design and fabrication of fibrous scaffolds for bone tissue engineering.

Original research article

by Jianghong Huang, Xiaoling Lei, Zhiwang Huang, Zhibin Rong, Haihang Li, Yixin Xie, Li Duan, Jianyi Xiong, Daping Wang, Shihui Zhu, Yujie Liang, Jianhao Wang, Jiang Xia

Artificial skins are biomaterials that can replace the lost skin or promote the regeneration of damaged skin. Skin regenerative biomaterials are highly applauded because they can exempt patients with severe burns from the painful procedure of autologous skin transplantation. Notwithstanding decades of research, biocompatible, degradable, and printable biomaterials that can effectively promote skin regeneration as a transplantation replacement in clinical use are still scarce. Here, we report one type of all-protein hydrogel material as the product of the enzymatic crosslinking reaction of gelatin and a recombinant type III collagen (rColIII) protein. Doping the rColIII protein in gelatin reduces the inflammatory response as an implant underneath the skin. The all-protein hydrogel can be bioprinted as scaffolds to support the growth and proliferation of 3T3 fibroblast cells. The hydrogel used as a wound dressing promotes wound healing in a rat model of skin damage, showing a faster and healthier recovery than the controls. The rColIII protein in the hydrogel has been shown to play a critical role in skin regeneration. Altogether, this work manifests the development of all-protein gelatin-rColIII hydrogel and demonstrates its use in wound healing. The gelatin-collagen hydrogel wound dressing thereby may become a promising treatment of severe wounds in the future.

Original research article

by Cristina Borràs-Novell, Mario García Causapié, Maria Murcia, Damien Djian, Óscar García-Algar


Non-invasive masks are designed based on generic facial models; therefore, difficulties in fitting patients’ unique characteristics are common. A poor fit of the mask may have consequences such as air leaks or pressure ulcers. It is possible to optimize the fit of interfaces by adapting them to a patient’s face. Our objective is to design an individualized silicone mask for non-invasive ventilation for a premature phantom using a three-dimensional (3D) scanner and bioprinter. The facial surface of the manikin was scanned with a 3D scanner in a supine position, in an incubator with a sliding mattress and in <2 min. We printed the tailor-made mask in 3 h with biocompatible and hypoallergenic silicone. When applied under a simulated clinical scenario, the mask possessed good structural reliability after post-processing and optimal mechanical features. We observed adequate thoracic excursion and 14% reduction in air leaks when the manikin was ventilated with the customized mask with a neonatal ventilator. We ink the edges of personalized and standard masks. After fitting them to phantoms, personalized mask showed better pressure distribution. Our subsequent research direction is to test the viability of personalizing non-invasive ventilation masks for very preterm infants of our department.

Original research article

by Xin Jiao, Xin Sun, Wentao Li, Wenxiang Chu, Yuxin Zhang, Yiming Li, Zengguang Wang, Xianhao Zhou, Jie Ma, Chen Xu, Kerong Dai, Jinwu Wang, Yaokai Gan

Bone defect is a serious orthopedic disease which has been studied for a long time. Alternative degradable biomaterials are required for bone repairing and regeneration to address the limitation of autogenous bone. β-tricalcium phosphate (β-TCP) is an alternative material with good cytocompatibility and has been used in bone defect treatment. However, whether β-TCP contributes to osteogenesis of bone marrow stem cells (BMSCs) through N6-methyladenosine (m6A) modification remains unknown. To address this issue, we verified the effects of β-TCP on osteogenesis of BMSCs. We also studied the expression of m6A-related enzymes in BMSCs after β-TCP treatment. Furthermore, the m6A level and stability of Runt-related transcription factor 2 (RUNX2) mRNA were investigated after β-TCP treatment. Finally, rat calvarial defect models were performed to detect expression level of osteogenic factors and m6A-related enzymes after the stimulation of three-dimension (3D)-printed β-TCP scaffolds. We found that β-TCP showed good biocompatibility and was osteoinductive. Meanwhile, methyltransferase-like 3 (METTL3) increased, causing the elevation of m6A level of RUNX2, results in stabler RUNX2 mRNA level. At last, based on the animal experiments, we demonstrated that the increase of RUNX2 and METTL3 levels was induced by β-TCP. These findings suggest that METTL3 increases the m6A level of RUNX2 mRNA after β-TCP induction, contributing to its stability, and the results in vivo also confirmed the osteogenic and bone-repair properties of β-TCP.

Original research article

by Rashik Chand, Beni Shimwa Muhire, Sanjairaj Vijayavenkataraman

Wall shear stress is the most critical factor in determining the viability of cells during the bioprinting process, and controlling wall shear stress remains a challenge in extrusion bioprinting. We investigated the effect of various bioprinting parameters using computational simulations on maximum wall shear stress (MWSS) in the nozzle to optimize the bioprinting process. Steady-state simulations were done for three nozzle geometries (conical, tapered conical, and cylindrical) with varying nozzle diameters (0.1 mm–0.5 mm) at different inlet pressure (0.025 MPa–0.25 MPa) as inlet conditions. Non-Newtonian power law was used to model the bioink rheology and four different bioinks with power-law constants ranging from 0.0863 to 0.5050 were examined. To capture the dynamic behavior of the bioink and the thread profile of the extruded bioink, transient simulations were carried out. Our results indicate that although the MWSS is lowest in the cylindrical nozzle, this stress condition lasts for a longer portion of the nozzle and for the same inlet pressure and nozzle diameter, the mass flow rate is lower compared to the tapered conical and conical nozzle, contributing to lower cell viability.

Original research article

by Yangyi Zhang, Jianfu Xia, Jiye Zhang, Jinlei Mao, Hao Chen, Hui Lin, Pan Jiang, Xinzhong He, Xiaodong Xu, Mingzhu Yin, Zhifei Wang

Rapid development of three-dimensional (3D) printing technique has enabled the production of many new materials for medical applications but the dry laboratory surgical training model made of soft and flexible materials is still insufficient. We established a new 3D-printed Nissen fundoplication training model of which materials simulate the real mechanical properties. In this study, 16 participants were divided into two groups: Experimental group and control group. The validity of model was tested using Likert scale by the experts and the experimental group. To evaluate the efficacy, performances of the experimental group were scored at the first, fourth, and eighth training by OSATS system and the duration of procedure was compared through the use of recorded video. Meanwhile, an ex vivo model was used to compare the performance of the experiment group and control group after the training in the same way. Our results showed that the 3D-printed model can support the future surgical applications, help improve surgical skills, and shorten procedure time after training.

Original research article

by Adam A. Mieloch, Julia A. Semba, Jakub Dalibor Rybka

At present, one of the main limitations of three-dimensional (3D) bioprinting in tissue engineering stems from a scarcity of biomaterials tailored for specific applications. Widely used hydrogels offer an optimal printability and a suitable environment for cell growth; however, they lack the mechanical strength required for non-soft tissues, for example, cartilage, tendons, and meniscus. This work investigated the physicochemical, mechanical, and biological characteristics of a 3D-printed polycaprolactone (PCL) reinforced with multiwalled carbon nanotubes (MWCNT) and “bamboo-like” carbon nanotubes (BCNT) with the following w/w % concentrations: 0.005%, 0.01%, 0.02%, and 0.2%. The materials were analyzed with subsequent techniques: Scanning electron microscopy, nanoindentation, parallel plate rheometry, and differential scanning calorimetry. Biological evaluations were performed with normal human articular chondrocytes by confocal microscopy and proliferation assay. The study revealed that the carbon nanotubes (CNT) addition improved the rheological properties of the material by increasing the setting temperature. Moderate enhancement was observed in terms of mechanical properties. The most significant difference was noted in cell adhesion and proliferation. Pure PCL did not facilitate cell growth and mainly apoptotic cells were observed on its surface. The addition of 0.01% MWCNT resulted in enhanced adhesion and proliferation; however, the morphology of the cells remained spherical, signifying a suboptimal surface for proliferation. Interestingly, PCL reinforced with 0.02% BCNT displayed excellent facilitation of cellular adhesion and proliferation, which is uncharacteristic of pure PCL. In summary, this study investigated the potential of CNT-reinforced PCL for 3D bioprinting and tissue engineering, highlighting key physicochemical, mechanical, and biological aspects of this biomaterial.

Original research article

by Chengjin Wang, Yang Yang, Jingyuan Ji, Yongcong Fang, Liliang Ouyang, Lei Zhang, Wei Sun
Intimal hyperplasia and restenosis caused by excessive proliferation of smooth muscle cells (SMC) are the main factors for the failure of stent implantation. Drug-eluting stents carried with antiproliferative drugs have emerged as a successful approach to alleviate early neointimal development. However, these agents have been reported to have an undesirable effect on re-endothelialization. In this study, we proposed an integrated bioresorbable stent coated with dipyridamole (DP)-loaded poly(D,L-lactide) (PDLLA) nanofibers. Three-dimensional (3D) bioresorbable stents were fabricated by printing on a rotation mandrel using polycaprolactone (PCL), and the stents were further coated with PDLLA/DP nanofibers. The in vitro degradation and drug release evaluation illustrated the potential for long-term release of DP. Stents coated with PDLLA/DP nanofibers showed excellent hemocompatibility. The cell viability, proliferation, and morphology analysis results revealed that stents coated with PDLLA/DP nanofibers could prevent the proliferation of SMC and have no adverse effects on endothelial cells. The in vivo implantation of stents coated with PDLLA/DP nanofibers showed initial patency and continuous endothelialization and alleviated neointimal formation. The attractive in vitro and in vivo performance indicated its potential for restenosis prevention and endothelialization.

Original research article

by Jihun Lee, Jaebum Sung, Jung Ki Jo, Hongyun So

This paper presents novel umbrella-shaped flexible devices to prevent vesicoureteral reflux along double-J stents, which is a backward flow of urine from the bladder to the kidney and is a critical issue in patients with urinary stones. The anti-reflux devices were designed to mechanically attach to the stent and were manufactured using three-dimensional (3D) printing and polymer casting methods. Based on the umbrella shapes, four different devices were manufactured, and the antireflux efficiency was demonstrated through in vitro experiments using a urination model. Consequently, penta-shaped devices exhibited the best anti-reflux performance (44% decrease in reflux compared to the stent without the device), and maximum efficiency occurred when the device was attached near the bladder-ureter junction. In addition, a disadvantage of 3D printing (i.e., unwanted rough surface) helped the device strongly adhere to the surface of the stent during the insertion operation. Finally, long-term soaking experiments revealed that the fabricated devices were mechanically robust and chemically stable (safe) even being soaked in urine for 4 weeks. The findings of this study support the use of additive manufacturing to make various flexible and biocompatible urological devices to mitigate critical issues in patients with urinary stones.

Original research article

by Peifang Dee, Sharlene Tan, Hortense Le Ferrand

Natural materials such as bone and enamel have intricate microstructures with inorganic minerals oriented to perform multiple mechanical and biological functions. Current additive manufacturing methods for biominerals from the calcium phosphate (CaP) family enable fabrication of custom-shaped bioactive scaffolds with controlled pore structures for patient-specific bone repair. Yet, these scaffolds do not feature intricate microstructures similar to those found in natural materials. In this work, we used direct material extrusion to 3D print water-based inks containing CaP microplatelets, and obtained microstructured scaffolds with various designs. To be shear-thinning and printable, the ink incorporated a concentration of 21 – 24 vol% CaP microplatelets of high aspect ratio. Good shape retention, print fidelity and overhanging layers were achieved by simultaneous printing and drying. Combined with the 3D design, versatile CaP microstructured objects can be built, from porous scaffolds to bulk parts. Extruded filaments featured a core-shell microstructure with graded microplatelet orientations, which was not affected by the printing parameters and the print design. A simple model is proposed to predict the core-shell microstructure according to the ink rheology. Given the remaining open porosity after calcination, microstructured scaffolds could be infiltrated with an organic phase in future to yield CaP biocomposites for hard tissue engineering.

Original research article

by Rong Li, Xuan Liu, Xin Yuan, Shanshan Wu, Li Li, Xuebing Jiang, Bo Li, Xian Jiang, Maling Gou
Hollow microneedle patches (HMNPs) have great promise for efficient and precise transdermal drug delivery in a painless manner. Currently, the clinical application of HMNPs is restricted by its complex manufacturing processes. Here, we use a new three-dimensional (3D) printing technology, static optical projection lithography (SOPL), for the fast fabrication of HMNPs. In this technology, a light beam is modulated into a customized pattern by a digital micromirror device (DMD) and projected to induce the spatial polymerization of monomer solutions which is controlled by the distribution of the light intensity in the monomer solutions. After an annulus picture is inputted into the DMD via the computer, the microneedles with hollow-cone structure can be precisely printed in seconds. By designing the printing pictures, the personalized HMNPs can be fast customized, which can afford the scale-up preparation of personalized HMNPs. Meanwhile, the obtained hollow microneedles (HMNs) have smooth surface without layer-by-layer structure in the commonly 3D-printed products. After being equipped with a micro-syringe, the HMNPs can efficiently deliver insulin into the skin by injection, resulting in effective control of the blood glucose level in diabetic mice. This work demonstrates a SOPL-based 3D printing technology for fast customization of HMNPs with promising medical applications.

Original research article

by Zoi Kanaki, Chrysoula Chandrinou, Ioanna-Maria Orfanou, Christina Kryou, Jill Ziesmer, Georgios A. Sotiriou, Apostolos Klinakis, Constantin Tamvakopoulos, Ioanna Zergioti

Cancer treatment with chemotherapeutic drugs remains to be challenging to the physician due to limitations associated with lack of efficacy or high toxicities. Typically, chemotherapeutic drugs are administered intravenously, leading to high drug concentrations that drive efficacy but also lead to known side effects. Delivery of drugs through transdermal microneedles (MNs) has become an important alternative treatment approach. Such delivery options are well suited for chemotherapeutic drugs in which sustained levels would be desirable. In the context of developing a novel approach, laser induced forward transfer (LIFT) was applied for bioprinting of gemcitabine (Gem) to coat polymethylmethacrylate MNs. Gem, a chemotherapeutic agent used to treat various types of cancer, is a good candidate for MN-assisted transdermal delivery to improve the pharmacokinetics of Gem while reducing efficiency limitations. LIFT bioprinting of Gem for coating of MNs with different drug amounts and successful transdermal delivery in mice is presented in this study. Our approach produced reproducible, accurate, and uniform coatings of the drug on MN arrays, and on in vivo transdermal application of the coated MNs in mice, dose-proportional concentrations of Gem in the plasma of mice was achieved. The developed approach may be extended to several chemotherapeutics and provide advantages for metronomic drug dosing.

Review article

by Yanfang Wang, Jiejie Wang, Ziyu Ji, Wei Yan, Hong Zhao, Wenhua Huang, Huan Liu

Three-dimensional (3D) bioprinting is an emerging technology that is widely used in regenerative medicine. With the continuous development of the technology, it has attracted great attention and demonstrated promising prospects in ophthalmologic applications. In this paper, we review the three main types of 3D bioprinting technologies: Vat polymerization based bioprinting, extrusion-based bioprinting, and jetting-based bioprinting. We also present in this review the analysis of the usage of both natural and synthesized hydrogels as well as the types of cells adopted for bioinks. Cornea and retina are the two main types of ocular tissues developed in bioprinting, while other device and implants were also developed for the ocular disease treatment. We also summarize the advantages and limitations as well as the future prospects of the current bioprinting technologies based on systematic reviews.

Short communication

by Jun Yi-Wu, Chih-Hua Hsieh, Zheng-Ying Lin


Present methods used in three-dimensional (3D) printing, such as selective laser sintering (SLS) and multijet fusion (MJF), have limited applications, especially in relation to the manufacturing of biomedical products. The speed of SLS printing is too low, and high-speed 3D printing technology with MJF uses carbon black particles as a fusing agent, which cannot be removed from the completed 3D printed products. Carbon black and high-energy lasers are not suitable for biomedical applications, especially human implants. A new high-speed 3D method is therefore required. In this study, we used hot oil droplets (175°C) as a new type of fusing agent to melt the biomaterial thermoplastic polyurethane (TPU) powder particles to define the print area. This method replaces lasers and the carbon black fusing agent in high-speed 3D printing technology and is more energy efficient. In addition, this method can be used to not only print on TPU, but also on other flexible materials.

Short communication

by Kevin Tröndle, Guilherme Miotto, Ludovica Rizzo, Roman Pichler, Fritz Koch, Peter Koltay, Roland Zengerle, Soeren S. Lienkamp, Sabrina Kartmann, Stefan Zimmermann

We used arrays of bioprinted renal epithelial cell spheroids for toxicity testing with cisplatin. The concentration dependent cell death rate was determined using a lactate dehydrogenase assay. Bioprinted spheroids showed enhanced sensitivity to the treatment in comparison to monolayers of the same cell type. The measured dose-response curves revealed an inhibitory concentration of the spheroids of IC50 = 9 ± 3 μM in contrast to the monolayers with IC50 = 17 ± 2 μM. Fluorescent labeling of a nephrotoxicity biomarker, kidney injury molecule 1 indicated an accumulation of the molecule in the central lumen of the spheroids. Finally, we tested an approach for an automatic readout of toxicity based on microscopic images with deep learning. Therefore, we created a dataset comprising images of single spheroids, with corresponding labels of the determined cell death rates for training. The algorithm was able to distinguish between three classes of no, mild, and severe treatment effects with a balanced accuracy of 78.7%.

Methods

by Changxi Liu, Jia Liu, Chengliang Yang, Yujin Tang, Zhengjie Lin, Long Li, Hai Liang, Weijie Lu, Liqiang Wang

Bioprinting is an emerging multidisciplinary technology for organ manufacturing, tissue repair, and drug screening. The manufacture of organs in a layer-by-layer manner is a characteristic of bioprinting technology, which can also determine the accuracy of constructs confined by the printing resolution. The lack of sufficient resolution will result in defect generation during the printing process and the inability to complete the manufacture of complex organs. A computer vision-based method is proposed in this study to detect the deviation of the printed helix from the reference trajectory and calculate the modified reference trajectory through error vector compensation. The new printing helix trajectory resulting from the modified reference trajectory error is significantly reduced compared with the original helix trajectory and the correction efficiency exceeded 90%.