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.

Advances in the differentiation of pluripotent stem cells into vascular cells

Blood vessels constitute a closed pipe system distributed throughout the body,transporting blood from the heart to other organs and delivering metabolic waste products back to the lungs and kidneys.Changes in blood vessels are related to many disorders like stroke,myocardial infarction,aneurysm,and diabetes,which are important causes of death worldwide.Translational research for new appro-aches to disease modeling and effective treatment is needed due to the huge socio-economic burden on healthcare systems.Although mice or rats have been widely used,applying data from animal studies to human-specific vascular physiology and pathology is difficult.The rise of induced pluripotent stem cells(iPSCs)provides a reliable in vitro resource for disease modeling,regenerative medicine,and drug discovery because they carry all human genetic information and have the ability to directionally differentiate into any type of human cells.This review summarizes the latest progress from the establishment of iPSCs,the strategies for differentiating iPSCs into vascular cells,and the in vivo trans-plantation of these vascular derivatives.It also introduces the application of these technologies in disease modeling,drug screening,and regenerative medicine.Additionally,the application of high-tech tools,such as omics analysis and high-throughput sequencing,in this field is reviewed.

Prevascularized Micro‑/Nano‑Sized Spheroid/Bead Aggregates for Vascular Tissue Engineering

Efficient strategies to promote microvascularization in vascular tissue engineering,a central priority in regenerative medicine,are still scarce;nano-and micro-sized aggregates and spheres or beads harboring primitive microvascular beds are promising methods in vascular tissue engineering.Capillaries are the smallest type and in numerous blood vessels,which are distributed densely in cardiovascular system.To mimic this microvascular network,specific cell components and proangiogenic factors are required.Herein,advanced biofabrication methods in microvascular engineering,including extrusion-based and droplet-based bioprinting,Kenzan,and biogripper approaches,are deliberated with emphasis on the newest works in prevascular nano-and micro-sized aggregates and microspheres/microbeads.

Cartilage oligomeric matrix protein enhances the vascularization of acellular nerves

Vascularization of acellular nerves has been shown to contribute to nerve bridging.In this study,we used a 10-mm sciatic nerve defect model in rats to determine whether cartilage oligomeric matrix protein enhances the vascularization of injured acellular nerves.The rat nerve defects were treated with acellular nerve grafting（control group） alone or acellular nerve grafting combined with intraperitoneal injection of cartilage oligomeric matrix protein（experimental group）.As shown through two-dimensional imaging,the vessels began to invade into the acellular nerve graft from both anastomotic ends at day 7 post-operation,and gradually covered the entire graft at day 21.The vascular density,vascular area,and the velocity of revascularization in the experimental group were all higher than those in the control group.These results indicate that cartilage oligomeric matrix protein enhances the vascularization of acellular nerves.

Construction of Oriented Structure in Inner Surface of Small-Diameter Artificial Blood Vessels:A Review

There is an urgent need for small-diameter artificial blood vessels in clinic.Physical,chemical and biological factors should be integrated to avoid thrombosis and intimal hyperplasia after implantation and to promote successful fabrication of small-diameter artificial blood vessels.From a physical perspective,the internal oriented structures of natural blood vessels plays an important role in guiding the directional growth of cells,improving the blood flow environment,and promoting the regeneration of vascular tissue.In this review,the effects of the oriented structures on cells,including endothelial cells(ECs),smooth muscle cells(SMCs)and stem cells,as well as the effect of the oriented structures on hemodynamics and vascular tissue remodeling and regeneration are introduced.Various forms of oriented structures(fibers,grooves,channels,etc.)and their construction methods are also reviewed.Conclusions and future perspectives are given.It is expected to give some references to relevant researches.

Gibberellin promotes cambium reestablishment during secondary vascular tissue regeneration after girdling in an auxin-dependent manner in Populus

Secondary vascular tissue(SVT)development and regeneration are regulated by phytohormones.In this study,we used an in vitro SVT regeneration system to demonstrate that gibberellin(GA)treatment significantly promotes auxin-induced cambium reestablishment.Altering GA content by overexpressing or knocking down ent-kaurene synthase(KS)affected secondary growth and SVT regeneration in poplar.The poplar DELLA gene GIBBERELLIC ACID INSENSITIVE(PtoGAI)is expressed in a specific pattern during secondary growth and cambium regeneration after girdling.Overexpression of PtoGAI disrupted poplar growth and inhibited cambium regeneration,and the inhibition of cambium regeneration could be partially restored by GA application.Further analysis of the PtaDR5:GUS transgenic plants,the localization of PIN-FORMED 1(PIN1)and the expression of auxin-related genes found that an additional GA treatment could enhance the auxin response as well as the expression of PIN1,which mediates auxin transport during SVT regeneration.Taken together,these findings suggest that GA promotes cambium regeneration by stimulating auxin signal transduction.

Advances in hydrogel-based vascularized tissues for tissue repair and drug screening

The construction of biomimetic vasculatures within the artificial tissue models or organs is highly required for conveying nutrients,oxygen,and waste products,for improving the survival of engineered tissues in vitro.In recent times,the remarkable progress in utilizing hydrogels and understanding vascular biology have enabled the creation of three-dimensional(3D)tissues and organs composed of highly complex vascular systems.In this review,we give an emphasis on the utilization of hydrogels and their advantages in the vascularization of tissues.Initially,the significance of vascular elements and the regeneration mechanisms of vascularization,including angiogenesis and vasculogenesis,are briefly introduced.Further,we highlight the importance and advantages of hydrogels as artificial microenvironments in fabricating vascularized tissues or organs,in terms of tunable physical properties,high similarity in physiological environments,and alternative shaping mechanisms,among others.Furthermore,we discuss the utilization of such hydrogels-based vascularized tissues in various applications,including tissue regeneration,drug screening,and organ-on-chips.Finally,we put forward the key challenges,including multifunctionalities of hydrogels,selection of suitable cell phenotype,sophisticated engineering techniques,and clinical translation behind the development of the tissues with complex vasculatures towards their future development.

High-resolution anatomical and spatial transcriptome analyses reveal two types of meristematic cell pools within the secondary vascular tissue of poplar stem

The secondary vascular tissue emanating from meristems is central to understanding how vascular plants such as forest trees evolve,grow,and regulate secondary radial growth.However,the overall molecular characterization of meristem origins and developmental trajectories from primary to secondary vascular tissues in woody tree stems is technically challenging.In this study,we combined high-resolution anatomic analysis with a spatial transcriptome(ST)technique to define features of meristematic cells in a developmental gradient from primary to secondary vascular tissues in poplar stems.The tissue-specific gene expression of meristems and derived vascular tissue types were accordingly mapped to specific anatomical domains.Pseudotime analyses were used to track the origins and changes of meristems throughout the development from primary to secondary vascular tissues.Surprisingly,two types of meristematic-like cell pools within secondary vascular tissues were inferred based on high-resolution microscopy combined with ST,and the results were confirmed by in situ hybridization of,transgenic trees,and single-cell sequencing.The rectangle shape procambium-like(PCL)cells develop from procambium meristematic cells and are located within the phloem domain to produce phloem cells,whereas fusiform shape cambium zone(CZ)meristematic cells develop from fusiform metacambium meristematic cells and are located inside the CZ to produce xylem cells.The gene expression atlas and transcriptional networks spanning the primary transition to secondary vascular tissues generated in this work provide new resources for studying the regulation of meristem activities and the evolution of vascular plants.A web server(https://pgx.zju.edu.cn/stRNAPal/)was also established to facilitate the use of ST RNA-seq data.

Noncoding RNAs and Their Potential Therapeutic Applications in Tissue Engineering

Tissue engineering is a relatively new but rapidly developing field in the medical sciences. Noncoding RNAs(ncRNAs) are functional RNA molecules without a protein-coding function; they can regulate cellular behavior and change the biological milieu of the tissue. The application of ncRNAs in tissue engineering is starting to attract increasing attention as a means of resolving a large number of unmet healthcare needs, although ncRNA-based approaches have not yet entered clinical practice. In-depth research on the regulation and delivery of ncRNAs may improve their application in tissue engineering.The aim of this review is: to outline essential ncRNAs that are related to tissue engineering for the repair and regeneration of nerve, skin, liver, vascular system, and muscle tissue; to discuss their regulation and delivery; and to anticipate their potential therapeutic applications.

ANAC005 is a membrane-associated transcription factor and regulates vascular development in Arabidopsis

Vascular tissues are very important for providing both mechanical strength and long-distance transport. The molecular mechanisms of regulation of vascular tissue develop- ment are still not fully understood. In this study we identified ANACoo5 as a membrane-associated NAC family transcription factor that regulates vascular tissue development. Reporter gene assays showed that ANACoo5 was expressed mainly in the vascular tissues, increased expression of ANACoo5 protein in transgenic Arabidopsis caused dwarf phenotype, reduced xylem differentiation, decreased lignin content, repression of a lignin biosynthetic gene and genes related to cambium and primary wall, but activation of genes related to the secondary wall. Expression of a dominant repressor fusion of ANACoo5 had overall the opposite effects on vascular tissue differ- entiation and lignin synthetic gene expression. The ANACoo5- GFP fusion protein was localized at the plasma membrane, whereas deletion of the last 20 amino acids, which are mostly basic, caused its nuclear localization. These results indicate that ANACoo5 is a cell membrane-associated transcription factor that inhibits xylem tissue development in Arabidopsis.

Novel magnetic silk fibroin scaffolds with delayed degradation for potential long-distance vascular repair

Although with the good biological properties,silk fibroin(SF)is immensely restrained in long-distance vascular defect repair due to its relatively fast degradation and inferior mechanical properties.It is necessary to construct a multifunctional composite scaffold based on SF.In this study,a novel magnetic SF scaffold(MSFCs)was prepared by an improved infiltration method.Compared with SF scaffold(SFC),MSFCs were found to have better crystallinity,magnetocaloric properties,and mechanical strength,which was ascribed to the rational introduction of iron-based magnetic nanoparticles(MNPs).Moreover,in vivo and in vitro experiments demonstrated that the degradation of MSFCs was significantly extended.The mechanism of delayed degradation was correlated with the dual effect that was the newly formed hydrogen bonds between SFC and MNPs and the complexing to tyrosine(Try)to inhibit hydrolase by internal iron atoms.Besides,theβ-crystallization of protein in MSFCs was increased with the rise of iron concentration,proving the beneficial effect after MNPS doped.Furthermore,although macrophages could phagocytose the released MNPs,it did not affect their function,and even a reasonable level might cause some cytokines to be upregulated.Finally,in vitro and in vivo studies demonstrated that MSFCs showed excellent biocompatibility and the growth promotion effect on CD34-labeled vascular endothelial cells(VECs).In conclusion,we confirm that the doping of MNPs can significantly reduce the degradation of SFC and thus provide an innovative perspective of multifunctional biocomposites for tissue engineering.

High-throughput drug screening models of mature adipose tissues which replicate the physiology of patients’Body Mass Index(BMI)

Obesity is a complex and incompletely understood disease,but current drug screening strategies mostly rely on immature in vitro adipose models which cannot recapitulate it properly.To address this issue,we developed a statistically validated high-throughput screening model by seeding human mature adipocytes from patients,encapsulated in physiological collagen microfibers.These drop tissues ensured the maintenance of adipocyte viability and functionality for controlling glucose and fatty acids uptake,as well as glycerol release.As such,patients’BMI and insulin sensitivity displayed a strong inverse correlation:the healthy adipocytes were associated with the highest insulin-induced glucose uptake,while insulin resistance was confirmed in the underweight and severely obese adipocytes.Insulin sensitivity recovery was possible with two type 2 diabetes treatments,rosiglitazone and melatonin.Finally,the addition of blood vasculature to the model seemed to more accurately recapitulate the in vivo physiology,with particular respect to leptin secretion metabolism.

Construction of tissue-engineered vascular grafts with enhanced patency by integrating heparin,cell-adhesive peptide,and carbon monoxide nanogenerators into acellular blood vessels

Small-diameter tissue-engineered vascular grafts(sdTEVGs)have garnered significant attention as a potential treatment modality for vascular bypass grafting and replacement therapy.However,the intimal hyperplasia and thrombosis are two major complications that impair graft patency during transplantation.To address this issue,we fabricated the covalent-organic framework(COF)-based carbon monoxide(CO)nanogenerator-and co-immobilized with LXW-7 peptide and heparin to establish a multifunctional surface on TEVGs constructed from acellular blood vessels for preventing thrombosis and stenosis.The cell-adhesive peptide LXW-7 could capture endothelial-forming cells(EFCs)to promote endothelialization,while the antithrombotic molecule heparin prevented thrombus formation.The reactive oxygen species(ROS)-triggered CO release suppressed the adhesion and activation of macrophages,leading to the reduction of ROS and inflammatory factors.As a result,the endothelial-to-mesenchymal transition(EndMT)triggered by inflammation was restricted,facilitating the maintenance of the homeostasis of the neo-endothelium and preventing pathological remodeling in TEVGs.When transplanted in vivo,these vascular grafts exhibited negligible intimal hyperplasia and remained patent for 3 months.This achievement provided a novel approach for constructing antithrombotic and anti-hyperplastic TEVGs.