Three-dimensional bioprinting of gelatin methacryloyl (GelMA)

The three-dimensional (3D)bioprinting technology has progressed tremendously over the past decade.By controlling the size, shape,and architecture of the bioprinted constructs,3D bioprinting allows for the fabrication of tissue/organ-like constructs with strong structural-functional similarity with their in vivo counterparts at high fidelity.The bioink,a blend of biomaterials and living cells possessing both high biocompatibility and printability,is a critical component of bioprinting.In particular, gelatin methacryloyl (GelMA)has shown its potential as a viable bioink material due to its suitable biocompatibility and readily tunable physicochemical properties.Current GelMA-based bioinks and relevant bioprinting strategies for GelMA bioprinting are briefly reviewed.

Engineering in vitro human tissue models through bio‑design and manufacturing

In the era of burgeoning breakthroughs around medical and biomedical technologies,personalizing our medicine still sounds like a dream yet to be realized.Take cancer as an example,while the pool of anti-cancer therapeutic agents is tremendously large,pinpointing a drug or a combination of drugs that would always work out well for a given patient,remains quite impossible—in fact,we are not even close to this ideal scenario.Such an incapability,of course,originates primarily from the overly complex,volumetrically structured and dynamic microenvironments in a patient’s tumor,meaning that the same tumor as seen on Day 1 might be entirely different than when seen again a month later.

Fabrication of paper-based devices for in vitro tissue modeling

Paper devices have recently attracted considerable attention as a class of cost-effective cell culture substrates for various biomedical applications.The paper biomaterial can be used to partially mimic the in vivo cell microenvironments mainly due to its natural three-dimensional characteristic.The paper-based devices provide precise control over their structures as well as cell distributions,allowing recapitulation of certain interactions between the cells and the extracellular matrix.These features have shown great potential for the development of normal and diseased human tissue models.In this review,we discuss the fabrication of paper-based devices for in vitro tissue modeling,as well as the applications of these devices toward drug screening and personalized medicine.It is believed that paper as a biomaterial will play an essential role in the field of tissue model engineering due to its unique performances,such as good biocompatibility,eco-friendliness,cost-effectiveness,and amenability to various biodesign and manufacturing needs.

Targeting Hypoxic Tumors with Hybrid Nanobullets for Oxygen-Independent Synergistic Photothermal and Thermodynamic Therapy

Hypoxia is a feature of solid tumors and it hinders the therapeutic efficacy of oxygen-dependent cancer treatment.Herein,we have developed all-organic oxygen-independent hybrid nanobullets ZPA@HA-ACVA-AZ for the“precise strike”of hypoxic tumors through the dual-targeting effects from surface-modified hyaluronic acid(HA)and hypoxia-dependent factor carbonic anhydrase IX(CA IX)-inhibitor acetazolamide(AZ).The core of nanobullets is the special zinc(II)phthalocyanine aggregates(ZPA)which could heat the tumor tissues upon 808-nm laser irradiation for photothermal therapy(PTT),along with the alkyl chain-functionalized thermally decomposable radical initiator ACVA-HDA on the side chain of HA for providing oxygen-independent alkyl radicals for ablating hypoxic cancer cells by thermodynamic therapy(TDT).The results provide important evidence that the combination of reverse hypoxia hallmarks CA IX as targets for inhibition by AZ and synergistic PTT/TDT possess incomparable therapeutic advantages over traditional(reactive oxygen species(ROS)-mediated)cancer treatment for suppressing the growth of both hypoxic tumors and their metastasis.

Bioprinting of Small-Diameter Blood Vessels

There has been an increasing demand for bioengineered blood vessels for utilization in both regenerative medicine and drug screening.However,the availability of a true bioengineered vascular graft remains limited.Three-dimensional(3D)bioprinting presents a potential approach for fabricating blood vessels or vascularized tissue constructs of various architectures and sizes for transplantation and regeneration.In this review,we summarize the basic biology of different blood vessels,as well as 3D bioprinting approaches and bioink designs that have been applied to fabricate vascular and vascularized tissue constructs,with a focus on small-diameter blood vessels.

Designs andmethodologies to recreate in vitro human gutmicrobiota models

The human gut microbiota is widely considered to be a metabolic organ hidden within our bodies,playing a crucial role in the host’s physiology.Several factors affect its composition,so a wide variety of microbes residing in the gut are present in the world population.Individual excessive imbalances in microbial composition are often associated with human disorders and pathologies,and new investigative strategies to gain insight into these pathologies and define pharmaceutical therapies for their treatment are needed.In vitro models of the human gut microbiota are commonly used to study microbial fermentation patterns,community composition,and host-microbe interactions.Bioreactors and microfluidic devices have been designed to culture microorganisms from the human gut microbiota in a dynamic environment in the presence or absence of eukaryotic cells to interact with.In this review,we will describe the overall elements required to create a functioning,reproducible,and accurate in vitro culture of the human gut microbiota.In addition,we will analyze some of the devices currently used to study fermentation processes and relationships between the human gut microbiota and host eukaryotic cells.

3D-bioprinted cholangiocarcinoma-on-a-chip model for evaluating drug responses

Cholangiocarcinoma(CCA)is characterized by heterogeneous mutations and a refractory nature.Thus,the development of a model for effective drug screening is urgently needed.As the established therapeutic testing models for CCA are often ineffective,we fabricated an enabling three-dimensional(3D)-bioprinted CCA-on-a-chip model that to a good extent resembled the multicellular microenvironment and the anatomical microstructure of the hepato-vascular-biliary system to perform high-content antitumor drug screening.Specifically,cholangiocytes,hepatocytes,and vascular endotheliocytes were employed for 3D bioprinting of the models,allowing for a high degree of spatial and tube-like microstructural control.Interestingly,it was possible to observe CCA cells attached to the surfaces of the gelatin methacryloyl(GelMA)hydrogelembedded microchannels and overgrown in a thickening manner,generating bile duct stenosis,which was expected to be analogous to the in vivo configuration.Over 4000 differentially expressed genes were detected in the CCA cells in our 3D coculture model compared to the traditional two-dimensional(2D)monoculture.Further screening revealed that the CCA cells grown in the 3D traditional model were more sensitive to the antitumoral prodrug than those in the 2D monoculture due to drug biotransformation by the neighboring functional hepatocytes.This study provides proof-of-concept validation of our bioprinted CCA-on-a-chip as a promising drug screening model for CCA treatment and paves the way for potential personalized medicine strategies for CCA patients in the future.

A hepatocellular carcinoma–bone metastasis-on-a-chip model for studying thymoquinone-loaded anticancer nanoparticles

We report the development of a metastasis-on-a-chip platform to model and track hepatocellular carcinoma(HCC)-bone metastasis and to analyze the inhibitory effect of an herb-based compound,thymoquinone(TQ),in hindering the migration of liver cancer cells into the bone compartment.The bioreactor consisted of two chambers,one accommodating encapsulated HepG2 cells and one bone-mimetic niche containing hydroxyapatite(HAp).Above these chambers,amicroporous membrane was placed to resemble the vascular barrier,where medium was circulated over the membrane.It was observed that the liver cancer cells proliferated inside the tumor microtissue and disseminated from the HCC chamber to the circulatory flow and eventually entered the bone chamber.The number of metastatic HepG2 cells to the bone compartment was remarkably higher in the presence of HAp in the hydrogel.TQ was then used as ametastasis-controlling agent in both free form and encapsulated nanoparticles,to analyze its suppressing effect on HCC metastasis.Results indicated that the nanoparticle-encapsulated TQ provided a longer period of inhibitory effect.In summary,HCC-bone metastasis-on-a-chip platform was demonstrated to model certain key aspects of the cancer metastasis process,hence corroborating the potential of enabling investigations on metastasis-associated biology as well as improved anti-metastatic drug screening.

Microfluidic bubble-generator enables digital light processing 3D printing of porous structures

Three-dimensional(3D)printing is an emerging technique that has shown promising success in engineering human tissues in recent years.Further development of vatphotopolymerization printing modalities has significantly enhanced the complexity level for 3D printing of various functional structures and components.Similarly,the development of microfluidic chip systems is an emerging research sector with promising medical applications.This work demonstrates the coupling of a digital light processing(DLP)printing procedure with a microfluidic chip system to produce size-tunable,3D-printable porosities with narrow pore size distributions within a gelatin methacryloyl(GelMA)hydrogel matrix.It is found that the generation of size-tunable gas bubbles trapped within an aqueous GelMA hydrogel-precursor can be controlled with high precision.Furthermore,the porosities are printed in two-dimensional(2D)as well as in 3D using the DLP printer.In addition,the cytocompatibility of the printed porous scaffolds is investigated using fibroblasts,where high cell viabilities as well as cell proliferation,spreading,and migration are confirmed.It is anticipated that the strategy is widely applicable in a range of application areas such as tissue engineering and regenerative medicine,among others.

Droplet 3D cryobioprinting for fabrication of free-standing and volumetric structures

Droplet-based bioprinting has shown remarkable potential in tissue engineering and regenerative medicine.However,it requires bioinks with low viscosities,which makes it challenging to create complex 3D structures and spatially pattern them with different materials.This study introduces a novel approach to bioprinting sophisti-cated volumetric objects by merging droplet-based bioprinting and cryobioprinting techniques.By leveraging the benefits of cryopreservation,we fabricated,for thefirst time,intricate,self-supporting cell-free or cell-laden structures with single or multiple materials in a simple droplet-based bioprinting process that is facilitated by depositing the droplets onto a cryoplate followed by crosslinking during revival.The feasibility of this approach is demonstrated by bioprinting several cell types,with cell viability increasing to 80%–90%after up to 2 or 3 weeks of culture.Furthermore,the applicational capabilities of this approach are showcased by bio-printing an endothelialized breast cancer model.The results indicate that merging droplet and cryogenic bioprinting complements current droplet-based bioprinting techniques and opens new avenues for the fabrication of volumetric objects with enhanced complexity and functionality,presenting exciting potential for biomedical applications.

Organoids in concert:engineering in vitro models toward enhanced fidelity

Organoids have emerged as a powerful platform for studying complex biological processes and diseases in vitro.However,most studies have focused on individual organoids,overlooking the inter-organ interactions in vivo and limiting the physiological relevance of the models.To address this limitation,the development of a multi-organoid system has gained considerable attention.This system aims to recapitulate inter-organ communication and enable the study of complex physiological processes.This review provides a comprehensive overview of the recent advancements in organoid engineering and the emerging strategies for constructing a multi-organoid system.First,we highlight the critical mechanical,structural,and biochemical factors involved in designing suitable materials for the growth of different organoids.Additionally,we discuss the incorporation of dynamic culture environments to enhance organoid culture and enable inter-organoid communication.Furthermore,we explore techniques for manipulating organoid morphogenesis and spatial positioning of organoids to establish effective inter-organoid communication networks.We summarize the achievements in utilizing organoids to recapitulate inter-organ communication in vitro,including assembloids and microfluidic multiorganoid platforms.Lastly,we discuss the existing challenges and opportunities in developing a multi-organoid system from its technical bottlenecks in scalability to its applications toward complex human diseases.

Photonic control of ligand nanospacing in self-assembly regulates stem cell fate

Extracellular matrix(ECM)undergoes dynamic inflation that dynamically changes ligand nanospacing but has not been explored.Here we utilize ECM-mimicking photocontrolled supramolecular ligand-tunable Azo^(+)self-assembly composed of azobenzene derivatives(Azo^(+))stacked via cation-πinteractions and stabilized with RGD ligand-bearing poly(acrylic acid).Near-infrared-upconverted-ultraviolet light induces cis-Azo^(+)-mediated inflation that suppresses cation-πinteractions,thereby inflating liganded self-assembly.This inflation increases nanospacing of“closely nanospaced”ligands from 1.8 nm to 2.6 nm and the surface area of liganded selfassembly that facilitate stem cell adhesion,mechanosensing,and differentiation both in vitro and in vivo,including the release of loaded molecules by destabilizing water bridges and hydrogen bonds between the Azo^(+)molecules and loaded molecules.Conversely,visible light induces trans-Azo^(+)formation that facilitates cation-πinteractions,thereby deflating self-assembly with“closely nanospaced”ligands that inhibits stem cell adhesion,mechanosensing,and differentiation.In stark contrast,when ligand nanospacing increases from 8.7 nm to 12.2 nm via the inflation of self-assembly,the surface area of“distantly nanospaced”ligands increases,thereby suppressing stem cell adhesion,mechanosensing,and differentiation.Long-term in vivo stability of self-assembly via real-time tracking and upconversion are verified.This tuning of ligand nanospacing can unravel dynamic ligand-cell interactions for stem cell-regulated tissue regeneration.

Bioinspired Andrias davidianus-Derived wound dressings for localized drug-elution

Local drug delivery has received increasing attention in recent years.However,the therapeutic efficacy of local delivery of drugs is still limited under certain scenarios,such as in the oral cavity or in wound beds after resection of tumors.In this study,we introduce a bioinspired adhesive hydrogel derived from the skin secretions of Andrias davidianus(SSAD)as a wound dressing for localized drug elution.The hydrogel was loaded with aminoguanidine or doxorubicin,and its controlled drug release and healing-promoting properties were verified in a diabetic rat palatal mucosal defect model and a C57BL/6 mouse melanoma-bearing model,respectively.The results showed that SSAD hydrogels with different pore sizes could release drugs in a controllable manner and accelerate wound healing.Transcriptome analyses of the palatal mucosa suggested that SSAD could significantly upregulate pathways linked to cell adhesion and extracellular matrix deposition and had the ability to recruit keratinocyte stem cells to defect sites.Taken together,these findings indicate that property-controllable SSAD hydrogels could be a promising biofunctional wound dressing for local drug delivery and promotion of wound healing.

Self-targeting visualizable hyaluronate nanogel for synchronized intracellular release of doxorubicin and cisplatin in combating multidrug-resistant breast cancer

Multidrug-resistance(MDR)featuring complicated and poorly defined mechanisms is a major obstacle to the success of cancer chemotherapy in the clinic.Compound nanoparticles comprising multiple cytostatics with different mechanisms of action are commonly developed to tackle the multifaceted nature of clinical MDR.However,the different pharmacokinetics and release profiles of various drugs result in inconsistent drug internalization and suboptimal drug synergy at the tumor sites.In the present study,a type of self-targeting hyaluronate(HA)nanogels((CDDPH)^ANG/DOX)to reverse drug resistance through the synchronized pharmacokinetics,intratumoral distribution,and intracellular release of topoisomerase II inhibitor doxorubicin(DOX)and DNA-crosslinking agent cisplatin(CDDP)is developed.With prolonged circulation time and enhanced intratumoral accumulation in vivo,(CDDP)^HANG/DOX shows efficient drug delivery into the drug-resistant MCF-7/ADR breast cancer cells and enhanced antitumor activity.Besides,fluorescence imaging of DOX combined with the micro-computed tomography(micro-CT)imaging of CDDP facilitates the visualization of this combination tumor chemotherapy.With visualizable synchronized drug delivery,the self-targeting in situ crosslinked nanoplatform may hold good potential in future clinical therapy of advanced cancers.

Organ-on-a-chip platforms for accelerating the evaluation of nanomedicine

Nanomedicine involves the use of engineered nanoscale materials in an extensive range of diagnostic and therapeutic applications and can be applied to the treatment of many diseases.Despite the rapid progress and tremendous potential of nanomedicine in the past decades,the clinical translational process is still quite slow,owing to the difficulty in understanding,evaluating,and predicting nanomaterial behaviors within the complex environment of human beings.Microfluidics-based organ-on-a-chip(Organ Chip)techniques offer a promising way to resolve these challenges.Sophisticatedly designed Organ Chip enable in vitro simulation of the in vivo microenvironments,thus providing robust platforms for evaluating nanomedicine.Herein,we review recent developments and achievements in Organ Chip models for nanomedicine evaluations,categorized into seven broad sections based on the target organ systems:respiratory,digestive,lymphatic,excretory,nervous,and vascular,as well as coverage on applications relating to cancer.We conclude by providing our perspectives on the challenges and potential future directions for applications of Organ Chip in nanomedicine.

Brain endothelial cell-derived extracellular vesicles with a mitochondria-targeting photosensitizer effectively treat glioblastoma by hijacking the blood-brain barrier

Glioblastoma(GBM)is the most aggressive malignant brain tumor and has a high mortality rate.Photodynamic therapy(PDT)has emerged as a promising approach for the treatment of malignant brain tumors.However,the use of PDT for the treatment of GBM has been limited by its low blood-brain barrier(BBB)permeability and lack of cancer-targeting ability.Herein,brain endothelial cell-derived extracellular vesicles(bEVs)were used as a biocompatible nanoplatform to transport photosensitizers into brain tumors across the BBB.To enhance PDT efficacy,the photosensitizer chlorin e6(Ce6)was linked to mitochondria-targeting triphenylphosphonium(TPP)and entrapped into bEVs.TPPconjugated Ce6(TPP-Ce6)selectively accumulated in the mitochondria,which rendered brain tumor cells more susceptible to reactive oxygen species-induced apoptosis under light irradiation.Moreover,the encapsulation of TPP-Ce6 into b EVs markedly improved the aqueous stability and cellular internalization of TPP-Ce6,leading to significantly enhanced PDT efficacy in U87MG GBM cells.An in vivo biodistribution study using orthotopic GBM-xenografted mice showed that b EVs containing TPP-Ce6[b EV(TPP-Ce6)]substantially accumulated in brain tumors after BBB penetration via transferrin receptor-mediated transcytosis.As such,b EV(TPP-Ce6)-mediated PDT considerably inhibited the growth of GBM without causing adverse systemic toxicity,suggesting that mitochondria are an effective target for photodynamic GBM therapy.

Functional Tough Hydrogels: Design, Processing, and Biomedical Applications

Hydrogels are high-water-content soft materials with widely tunable physicochemical properties,resembling soft tissues.Tremendous progress in engineering hydrogels with good biocompatibility,suitable bioactivities,and controlled geometries has made them promising candidates for broad applications.Nevertheless,conventional hydrogels usually suffer from weak mechanical properties,limiting their use in biomedical settings involving load-bearing and persistent mechanical deformations.Inspired by the extreme mechanical properties and multiscale hierarchical structures of biological tissues,mechanically robust tough hydrogels have been developed.Combining robust mechanical properties and other desired performance characteristics in functional tough hydrogels expands their opportunities in biomedical fields.This Account seeks to guide the readership regarding the recent progress in functional tough hydrogels with a focus on molecular/structural design and novel fabrications,particularly surrounding the works reported by our groups.Meanwhile,functional tough hydrogels for multiple biomedical applications are discussed,highlighting the underlying mechanisms governing their relevant applications.We begin by introducing the definition,measurements,and design principles of tough hydrogels and hydrogel adhesives in terms of soft materials mechanics.Various molecular and structural engineering approaches by building mechanical dissipation into stretchable hydrogels to realize stress homogenization or energy dissipation are exploited to fabricate tough hydrogels.Molecular engineering-based network architecture design of homogeneous hydrogels and structural engineering-based design of heterogeneous hydrogels are elaborated.The conventional energy-dissipation-based tough hydrogels are reinforced by the sacrificial bonds or components,leading to a substantial toughness reduction in subsequent loading cycles.To this end,new molecular designs,including highly entangled hydrogels and sliding-ring hydrogels,have been developed to resolve the toughness−hysteresis conflict.In addition,novel processing techniques,including salting out,freeze casting,and three-dimensional(bio)printing,are exploited to manipulate the multiscale structures and geometries for tough hydrogel fabrication.As some of the most actively studied materials in recent years,functional tough hydrogels are finding promising applications as bioadhesives/coatings,tissue-engineering scaffolds,soft robot/actuators,and bioelectronics interfaces.The development of tough bioadhesives/coatings lies in constructing strong interfacial linkages between the tough hydrogels and the underlying substrates,having broad applications in wound closure and drug delivery.Tough hydrogels have also been widely studied for use in tissue engineering and regenerative medicine,although the conflict of mechanical robustness−cellular function restricts their practical applications.The flexible and compliant tough hydrogels with stimuli-responsive shape shifting and pressure-triggered actuation make them good candidates as actuators and soft robots for biomedical devices dealing with soft tissues.Conductive tough hydrogels also have been widely exploited for utility in bioelectronics.In the end,we highlight the major challenges and emphasize the trends in developing the next-generation functional tough hydrogels for practical biomedical and medical applications.

Microarchitectural mimicking of stroma-induced vasculature compression in pancreatic tumors using a 3D engineered model

Fibrotic tumors,such as pancreatic ductal adenocarcinoma(PDAC),are characterized for high desmoplastic reaction,which results in high intra-tumoral solid stress leading to the compression of blood vessels.These microarchitectural alterations cause loss of blood flow and poor intra-tumoral delivery of therapeutics.Currently,there is a lack of relevant in vitro models capable of replicating these mechanical characteristics and to test anti-desmoplastic compounds.Here,a multi-layered vascularized 3D PDAC model consisting of primary human pancreatic stellate cells(PSCs)embedded in collagen/fibrinogen(Col/Fib),mimicking tumor tissue within adjunct healthy tissue,is presented to study the fibrosis-induced compression of vasculature in PDAC.It is demonstrated how the mechanical and biological stimulation induce PSC activation,extracellular matrix production and eventually vessel compression.The clinical relevance is confirmed by correlating with patient transcriptomic data.Furthermore,the effects of gradual vessel compression on the fluid dynamics occurring within the channel is evaluated in silico.Finally,it is demonstrated how cancer-associated fibroblast(CAF)-modulatory therapeutics can inhibit the cell-mediated compression of blood vessels in PDAC in vitro,in silico and in vivo.It is envisioned that this 3D model is used to improve the understanding of mechanical characteristics in tumors and for evaluating novel anti-desmoplastic therapeutics.

Biomedical applications of magnetosomes: State of the art and perspectives

Magnetosomes, synthesized by magnetotactic bacteria (MTB), have been used in nano- and biotechnological applications, owing to their unique properties such as superparamagnetism, uniform size distribution, excellent bioavailability, and easily modifiable functional groups. In this review, we first discuss the mechanisms of magnetosome formation and describe various modification methods. Subsequently, we focus on presenting the biomedical advancements of bacterial magnetosomes in biomedical imaging, drug delivery, anticancer therapy, biosensor. Finally, we discuss future applications and challenges. This review summarizes the application of magnetosomes in the biomedical field, highlighting the latest advancements and exploring the future development of magnetosomes.

3D bioprinted organ-on-chips

Organ-on-a-chip(OOC)platforms recapitulate human in vivo-like conditions more realistically compared to many animal models and conventional two-dimensional cell cultures.OOC setups benefit from continuous perfusion of cell cultures through microfluidic channels,which promotes cell viability and activities.Moreover,microfluidic chips allow the integration of biosensors for real-time monitoring and analysis of cell interactions and responses to administered drugs.Three-dimensional(3D)bioprinting enables the fabrication of multicell OOC platforms with sophis-ticated 3D structures that more closely mimic human tissues.3D-bioprinted OOC platforms are promising tools for understanding the functions of organs,disruptive influences of diseases on organ functionality,and screening the efficacy as well as toxicity of drugs on organs.Here,common 3D bioprinting techniques,advantages,and limitations of each method are reviewed.Additionally,recent advances,applica-tions,and potentials of 3D-bioprinted OOC platforms for emulating various human organs are presented.Last,current challenges and future perspectives of OOC plat-forms are discussed.