Engineering vascularized organotypic tissues via module assembly

Adequate vascularization is a critical determinant for the successful construction and clinical implementation of complex organotypic tissue models. Currently, low cell and vessel density and insufficient vascular maturation make vascularized organotypic tissue construction difficult,greatly limiting its use in tissue engineering and regenerative medicine. To address these limitations, recent studies have adopted pre-vascularized microtissue assembly for the rapid generation of functional tissue analogs with dense vascular networks and high cell density. In this article, we summarize the development of module assembly-based vascularized organotypic tissue construction and its application in tissue repair and regeneration, organ-scale tissue biomanufacturing, as well as advanced tissue modeling.

Bio-Manufacturing Research Center at Tsinghua University

Overview and research infrastructure As one of the leading laboratories in the interdisciplinary field of additive manufacturing and bio-3D printing,the Bio-Manufacturing Center at Tsinghua University is highly dedicated to conducting cutting-edge research in the emerging field of bio-manufacturing.The latter is comprised of research involving biomaterials,living cells,proteins,and/or other biological compounds as basic building blocks to fabricate biomimetic structures,in vitro functional biological models and/or cellular systems with application to tissue engineering,regenerative medicine,disease pathogenesis,drug screening,and tissue/organ-on-a-chip.

Three-dimensional printing: review of application in medicine and hepatic surgery

Three-dimensional(3D) printing(3DP) is a rapid prototyping technology that has gained increasing recognition in many different fields. Inherent accuracy and low-cost property enable applicability of 3DP in many areas, such as manufacturing, aerospace,medical, and industrial design. Recently, 3DP has gained considerable attention in the medical field. The image data can be quickly turned into physical objects by using 3DP technology. These objects are being used across a variety of surgical specialties. The shortage of cadaver specimens is a major problem in medical education. However, this concern has been solved with the emergence of 3DP model. Custom-made items can be produced by using 3DP technology. This innovation allows 3DP use in preoperative planning and surgical training. Learning is difficult among medical students because of the complex anatomical structures of the liver. Thus, 3D visualization is a useful tool in anatomy teaching and hepatic surgical training. However,conventional models do not capture haptic qualities. 3DP can produce highly accurate and complex physical models. Many types of human or animal differentiated cells can be printed successfully with the development of 3D bio-printing technology. This progress represents a valuable breakthrough that exhibits many potential uses, such as research on drug metabolism or liver disease mechanism. This technology can also be used to solve shortage of organs for transplant in the future.

Design, Characterization, and 3D Printing of Cardiovascular Stents with Zero Poisson’s Ratio in Longitudinal Deformation

Inherent drawbacks associated with drug-eluting stents have prompted the development of bioresorbable cardiovascular stents.Additive manufacturing(3-dimentional(3D)printing)has been widely applied in medical devices.In this study,we develop a novel screw extrusion-based 3D printing system with a new designed mini-screw extruder to fabricate stents.A stent with a zero Poisson’s ratio(ZPR)structure is designed,and a preliminary monofilament test is conducted to investigate appropriate fabrication parameters.3D-printed stents with different geometric structures are fabricated and analyzed by observation of the surface morphology.An evaluation of the mechanical properties and a preliminary biological evaluation of 3D-printed stents with different parameters are carried out.In conclusion,the screw extrusion-based 3D printing system shows potential for customizable stent fabrication.

Medical Additive Manufacturing: From a Frontier Technology to the Research and Development of Products

1.Research and development(R&D)and the challenges of raw materials for medical additive manufacturing Raw materials for medical additive manufacturing have a wide range of commonalities that are also seen in many other fields,making them an important basis in the field of three-dimensional(3D)printing.Problems and challenges related to material types,powder properties,formability,viscoelasticity,and so forth also share common features.For example,many metal materials are used in the field of aviation,while metals,polymers,and inorganic materials are used in the field of biomedicine.The most widely used materials in biomedicine are biocompatible.Various homogeneous and non-homogeneous composites are also available for 3D printing,and impose an additional challenge in additive manufacturing;the use of heterogeneous composites in 3D printing is particularly challenging.

Oriented cellulose hydrogel:Directed tissue regeneration for reducing corneal leukoplakia and managing fungal corneal ulcers

Fungal corneal ulcer is one of the leading causes of corneal blindness in developing countries.Corneal scars such as leukoplakia are formed due to inflammation,oxidative stress and non-directed repair,which seriously affect the patients'subsequent visual and life quality.In this study,drawing inspiration from the oriented structure of collagen fibers within the corneal stroma,we first proposed the directional arrangement of CuTA-CMHT hydrogel system at micro and macro scales based on the 3D printing extrusion method combined with secondary patterning.It played an antifungal role and induced oriented repair in therapy of fungal corneal ulcer.The results showed that it effectively inhibited Candida albicans,Aspergillus Niger,Fusarium sapropelum,which mainly affects TNF,NF-kappa B,and HIF-1 signaling pathways,achieving effective antifungal functions.More importantly,the fibroblasts interacted with extracellular matrix(ECM)of corneal stroma through formation of focal adhesions,promoted the proliferation and directional migration of cells in vitro,induced the directional alignment of collagen fibers and corneal stromal orthogonally oriented repair in vivo.This process is mainly associated with MYLK,MYL9,and ITGA3 molecules.Furthermore,the downregulation the growth factors TGF-βand PDGF-βinhibits myofibroblast development and reduces scar-type ECM production,thereby reducing corneal leukoplakia.It also activates the PI3K-AKT signaling pathway,promoting corneal healing.In conclusion,the oriented CuTA-CMHT hydrogel system mimics the orthogonal arrangement of collagen fibers,inhibits inflammation,eliminates reactive oxygen species,and reduces corneal leukoplakia,which is of great significance in the treatment of fungal corneal ulcer and is expected to write a new chapter in corneal tissue engineering.

Culture models produced via biomanufacturing for neural tissue-like constructs based on primary neural and neural stem cells

Neural tissue-like constructs have important application potential in both neural tissue regeneration and individual medical treatment due to the ideal bioenvironment they provide for the growth of primary and stem cells.The biomaterials used in threedimensional(3D)biomanufacturing techniques play a critical role in bioenvironment fabrication.They help optimize the manufacturing techniques and the long-term environment that supports cell structure and nutrient transmission.This paper reviews the current progress being made in the biomaterials utilized in neural cell cultures for in vitro bioenvironment construction.The following four requirements for biomaterials are evaluated:biocompatibility,porosity,supportability,and permeability.This study also summarizes the recent culture models based on primary neural cells.Furthermore,the biomaterials used for neural stem cell constructs are discussed.This study’s results indicate that compared with traditional twodimensional(2D)cultures(with minimal biomaterial requirements),modulus 3D cultures greatly benefit from optimized biomaterials for long-term culturing.

3D magnetic field guided sunflower-like nanocatalytic active swarm targeting patients-derived organoids

Nanocatalytic medicine triggering in situ catalytic reactions has been considered as a promising strategy for tumor-selective therapeutics.However,the targeted distribution of nanocatalysts was still low,considering the absence of targeting propulsion capability.Here,encouraged by the fast-developing controllable microrobotics for targeting delivery,a sunflower-like nanocatalytic active swarm(SNCAS)controlled by a three-dimensional(3D)magnetic field was proposed for synergistic tumorselective and magnetic-actively tumor-targeting therapeutics.Furthermore,a patient-derived renal cancer cell 3D organoid was utilized for the verification of the effective tumor therapeutic outcomes.Under the targeted control of 3D magnetic field,the multiple cascade catalytic efficiency of SNCAS based on Fenton reaction was evaluated,resulting in efficient tumor cell apoptosis and death.For the patient-derived organoid treatment,the SNCAS presented significant lethality toward 3D organoid structure to induce cell apoptosis with the collapse of organoid morphology.The targeting efficiency was further enhanced under the magnetic-controllable of SNCAS.Overall,empowered by the magnetic control technology,the synergistic therapeutic strategy based on controllable swarm combined active targeting and tumor-specific catalytic nanomedicine has provided a novel way for advanced cancer therapy.Meanwhile,3D patient-derived organoids were proved as a powerful tool for the effectiveness verification of nanocatalytic medicine.

Digital light processing (DLP)-based (bio)printing strategies for tissue modeling and regeneration

Digital light processing(DLP)-based bioprinting technology has recently aroused considerable concerns as a strategy to deliver biomedical materials and/or specific cells to create sophisticated structures for various tissue modeling and regeneration.In this review,we display a concise introduction of DLP bioprinting,and a further discussion on the design and manufacture of DLP(bio)printer with varied bioinks and their biomedical applications toward drug screening,disease modeling,tissue repair,and regenerative medicine.Finally,the advantages,challenges,and perspectives of the DLP printing platforms are detailed.It is believed that DLP bioprinting will play a decisive role in the field of tissue model and regenerative medicine,mainly due to its time-efficient,higher resolution,and amenability to automation for various tissue needs.

Creating a semi-opened micro-cavity ovary through sacrificial microspheres as an in vitro model for discovering the potential effect of ovarian toxic agents

The bio-engineered ovary is an essential technology for treating female infertility.Especially the development of relevant in vitro models could be a critical step in a drug study.Herein,we develop a semi-opened culturing system(SOCS)strategy that maintains a 3D structure of follicles during the culture.Based on the SOCS,we further developed micro-cavity ovary(MCO)with mouse follicles by the microsphere-templated technique,where sacrificial gelatin microspheres were mixed with photo-crosslinkable gelatin methacryloyl(GelMA)to engineer a micro-cavity niche for follicle growth.The semi-opened MCO could support the follicle growing to the antral stage,secreting hormones,and ovulating cumulus-oocyte complex out of the MCO without extra manipulation.The MCO-ovulated oocyte exhibits a highly similar transcriptome to the in vivo counterpart(correlation of 0.97)and can be fertilized.Moreover,we found that a high ROS level could affect the cumulus expansion,which may result in anovulation disorder.The damage could be rescued by melatonin,but the end of cumulus expansion was 3h earlier than anticipation,validating that MCO has the potential for investigating ovarian toxic agents in vitro.We provide a novel approach for building an in vitro ovarian model to recapitulate ovarian functions and test chemical toxicity,suggesting it has the potential for clinical research in the future.

Towards smart scanning probe lithography: a framework accelerating nano-fabrication process with in-situ characterization via machine learning

Scanning probe lithography(SPL)is a promising technology to fabricate high-resolution,customized and costeffective features at the nanoscale.However,the quality of nano-fabrication,particularly the critical dimension,is significantly influenced by various SPL fabrication techniques and their corresponding process parameters.Meanwhile,the identification and measurement of nano-fabrication features are very time-consuming and subjective.To tackle these challenges,we propose a novel framework for process parameter optimization and feature segmentation of SPL via machine learning(ML).Different from traditional SPL techniques that rely on manual labeling-based experimental methods,the proposed framework intelligently extracts reliable and global information for statistical analysis to finetune and optimize process parameters.Based on the proposed framework,we realized the processing of smaller critical dimensions through the optimization of process parameters,and performed direct-write nano-lithography on a large scale.Furthermore,data-driven feature extraction and analysis could potentially provide guidance for other characterization methods and fabrication quality optimization.

Advances in 3D Bioprinting

Three-dimensional(3D)bioprinting has emerged as a promising approach for engineering functional tissues and organs by layer-by-layer precise positioning of biological materials,living cells,and biochemical components.Compared with nonbiological printing,3D bioprinting involves additional complexities and technical challenges owing to the processing of living cells,such as the appropriate biomaterials that fulfill the requirements for both printability and functionality.In this review,we first introduce the development course of 3D bioprinting,highlighting innovative forms of living building blocks and advances in enabling techniques of 3D bioprinting.We then summarize the state-of-the-art advancements in 3D bioprinting for biomedical applications,including macroscale tissue or organ bioprinting,disease modeling,microphysiological systems,biobots,and bioprinting in space.Despite the rapid development of 3D bioprinting over the past decades,most 3D bioprinted tissue or organ constructs are still far from being suitable for clinical translation,and it is necessary for the field of bioprinting to shift its focus from shape mimicking towards functionality development.Therefore,we provide our perspectives on this burgeoning field with an emphasis on functional maturation post printing and translational applications at the bedside.

Coaxial Embedded Printing of Gelatin Methacryloyl-alginate Double Network Hydrogel for Multilayer Vascular Tubes

The reconstruction of vascular-like tissues exhibiting a typical three-layer structure in vitro is vital to bio-fabrication research.It enables the realization of more complicated micro-environments,such as myocardium,liver,and tumor,which enables us to investigate their specific physiological phenomena or pathological mecha-nisms.Herein,we propose a coaxial embedded printing method,where the gelatin methacrylate(GelMA)-alginate composite hydrogel and sacrificial materials are extruded from a coaxial nozzle into a cylinder mold.By applying this method,we achieve the rapid fabrication of multilayer tube structures with inner diameters ranging from 400 to 1000μm.In addition,myoblasts are encapsulated in the hydrogel,and the cells show high viability.Moreover,we encapsulate smooth muscle cells(SMCs)and the human umbilical vein endothelial cells-T1(HUVEC-T1)cell line in the hydrogel to form vascular-like tissues,and the cells exhibit good morphology and protein expression.These results suggest that a vascular tube fabricated using the proposed method can serve as a vascular model for in vitro studies.

Antioxidant activity of phlorotannins from brown algae

The antioxidant activity of the phlorotannins extracted from five marine algaespecies(Saccharina latissima,Alaria esculenta,Laminaria digitata,Fucus vesiculosusand Ascophyllum nodosum)was studied.Three phlorotannin groups,including soluble,membrane-bound,and extracted membrane-bound phlorotannins obtained by two solvent extraction methods were investigated for their DPPH radical scavenging activity.F.vesiculosusand A.nodosumshowed the highest phlorotannin yield(14.83 mg-extract/g-algae and 12.80 mg-extract/g-algae,respectively)among the five algaespecies.Their soluble phlorophannin(SP),membrane-bound phlorotannin(MP)and extracted membrane-bound phlorotannin(eMP)extracts all showed equal or greater DPPH radical scavenging activity than the commercial antioxidants of butylated hydroxytoluene and ascorbic acid.The antioxidant potential that combines phlorotannin yield and antioxidant activity of the MP extracts of F.vesiculosusand A.nodosum(5890mL/g and 5278 mL/g algae,respectively)were higher than those of SP and eMP,suggesting that the MPs of F.vesiculosusand A.nodosumhad great potential to be used as antioxidants.Different extraction methods also showed significantly different effects on the antioxidant activity of the phlorotannin extracts.

Construction and Application of in vitro Alveolar Models Based on 3D Printing Technology

Increasing lung diseases,mutating coronaviruses,and the development of new compounds urgently require biomimetic in vitro lung models for lung pathology,toxicology,and pharmacology.The current construction strategies for lung models mainly include animal models,2D cell culture,lung-on-a-chip,and lung organoids.However,current models face difficulties in reproducing in vivo-like alveolar size and vesicle-like structures,and are unable to contain multiple cell types.In this study,a strategy for constructing alveolar models based on degradable hydrogel microspheres is proposed.Hydrogel microspheres,200-250μm in diameter,were prepared using a self-developed printing technique driven by alternating viscous and inertial forces.Microcapsules were further constructed using a coacervation-based layer-by-layer technique and core liquefaction.Three types of cells were inoculated and co-cultured on hydrogel capsules based on optimized microcapsule surface treatment strategies.Finally,an in vitro three-dimensional endothelial alveolar model with a multicellular composition and vesicle-like structure with a diameter of approximately 230μm was successfully constructed.Cells in the constructed alveolar model maintained a high survival rate.The LD_(50)values of glutaraldehyde based on the constructed models were in good agreement with the reference values,validating the potential of the model for future toxicant and drug detection.