An oxygenating colloidal bioink for the engineering of biomimetic tissue constructs

Ensuring a sufficient oxygen supply is pivotal for the success of bioprinting applications since it fosters tissue integration and natural regeneration.Variation in oxygen concentration among diverse tissues necessitates the precise recreation of tissue-specific oxygen levels in imprinted constructs to support the survival of targeted cells.Although oxygen-releasing biomaterials,such as oxygen-generating microparticles(OMPs),have shown promise for enhancing the oxygen supply of microenvironments in injured tissues,whether this approach is scalable for large tissues and whether tissue-specific bioinks with varying OMP concentrations remain printable remain unknown.This study addresses this critical gap by introducing an innovative class of engineered oxygenated bioinks that combine colloidal-based microgels with OMPs.We report that incorporating nanosized calcium peroxide(nCaO_(2))and manganese oxide nanosheets(nMnO_(2))into hydrophobic polymeric microparticles enables precise modulation of oxygen release while controlling hydrogen peroxide release.Moreover,the fabrication of oxygenating and cytocompatible colloidal gels is achieved using an aqueous two-phase system.This study thoroughly evaluates the fundamental characteristics of the resulting bioink,including its rheological behaviors,printability,shape fidelity,mechanical properties,and oxygen release properties.Moreover,this study demonstrates the macroscopic scalability and cytocompatibility of printed constructs produced via cell-laden oxygenating colloidal bioinks.By showcasing the effectiveness of extrusion-based bioprinting,this study underscores how it can be used to fabricate biomimetic tissues,indicating its potential for new applications.The findings presented here advance the bioprinting field by achieving scalability with both high cell viability and the possibility of mimicking specifically oxygenated tissues.This work thereby offers a promising avenue for the development of functional tissues with enhanced physiological relevance.

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.

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.

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.

From Bench to the Clinic: The Path to Translation of Nanotechnology-Enabled mRNA SARS-CoV-2 Vaccines

During the last decades,the use of nanotechnology in med icine has effectively been translated to the design of drug delivery systems,nanostructured tissues,diagnostic platforms,and novel nanomaterials against several human diseases and infectious pathogens.Nanotechnology-enabled vaccines have been positioned as solutions to mitigate the pandemic outbreak caused by the novel pathogen severe acute respiratory syndrome coronavirus 2.To fast-track the development of vaccines,unprecedented industrial and academic collaborations emerged around the world,resulting in the clinical translation of effective vaccines in less than one year.In this article,we provide an overview of the path to translation from the bench to the clinic of nanotechnology-enabled messenger ribonucleic acid vaccines and examine in detail the types of delivery systems used,their mechanisms of action,obtained results during each phase of their clinical development and their regulatory approval process.We also analyze how nanotechnology is impacting global health and economy during the COVID-19 pandemic and beyond.

A 3D in vitro co-culture model for evaluating biomaterial-mediated modulation of foreign-body responses

The immune response after implantation of a biomaterial may shorten the functional life of the implant,depending on the degree of the response.In this study,we used a polyacrylamide-alginate(PAAm-Alg)hydrogel,which has been previously characterized as a biocompatible material and shown to enhance regeneration of cartilage in vivo,along with a graphiteenhanced hydrogel(PAAm-Alg-G)as a non-biocompatible counterpart,to evaluate macrophage attachment and polarization to pro-or anti-inflammatory phenotypes.The performance of each biomaterial in the presence of fibroblasts and chondrocytes was validated by an in vitro model which demonstrated modulation of the foreign-body response.A blend of 5%gelatin methacryloyl and 0.1%methacrylated hyaluronic acid was optimized to mimic the extracellular matrix(ECM)and support cell viability,proliferation,migration,and functionality at an initial concentration of 3.25×10^(5) cells/mL.The PAAm-Alg-G hydrogel localized in the simulated ECM showed cytotoxic and genotoxic effects for both fibroblasts and chondrocytes,while exhibiting a proliferative effect on macrophages with elevated immune response.The M1/M2 ratio was 0.73 for PAAm-Alg hydrogel but 2.64 for PAAm-Alg-G,leading to significant M1 dominance(p<0.0001),as expected,on day 13.Moreover,loading PAAm-Alg hydrogel with transforming growth factor beta-3(TGF-β3)resulted in a slightly more balanced M1/M2 ratio of 0.87(p>0.05).The interleukin-6(IL-6)concentration secreted in the presence of PAAm-Alg hydrogel(4.58 pg/mL)significantly decreased(p<0.0001)on day 13,while the increase(p<0.0001)in interleukin-10(IL-10)concentration(120.73 pg/mL)confirmed the switch from a pro-inflammatory to an anti-inflammatory response.Predicting immune responses by developing a simplistic yet powerful three-dimensional in vitro model provides advantages in preparing for clinical use of biomaterials.

In vitro 3D malignant melanoma model for the evaluation of hypericin-loaded oil-in-water microemulsion in photodynamic therapy

Advances in biomimetic three-dimensional(3D) melanoma models have brought new prospects of drug screening and disease modeling, since their physiological relevancy for recapitulating in vivo tumor architectures is more accurate than traditional two-dimensional(2D) cell culture. Gelatin methacryloyl(GelMA) is widely used as a tissue-engineered scaffold hydrogel for 3D cell culture. In the present study, an in vitro 3D malignant melanoma model based on Gel MA was fabricated to evaluate the efficiency of hypericin(Hy)-loaded microemulsion(ME) in photodynamic therapy against melanoma. The ME was produced by the spontaneous emulsification method to enhance the bioavailability of Hy at tumor sites. Hy-loaded MEs were applied to a 3D malignant melanoma model made using 6% Gel MA and the co-culture of B16F10 and Balb/c 3T3 cells,followed by crosslinking using violet light(403 nm). The observation revealed excellent cell viability and the presence of F-actin cytoskeleton network. Hy-loaded MEs exhibited higher phototoxicity and cell accumulation(about threefold) than free Hy, and the cells cultured in the 3D system displayed lower susceptibility(about 2.5-fold) than those in 2D culture.These findings indicate that the developed MEs are potential delivery carriers for Hy;furthermore, Gel MA hydrogel-based modeling in polydimethylsiloxane(PDMS) molds is a user-friendly and cost-effective in vitro platform to investigate drug penetration and provide a basis for evaluating nanocarrier efficiency for skin cancer and other skin-related diseases.

Engineering nanomedicines for immunogenic eradication of cancer cells:Recent trends and synergistic approaches

Resistance to cancer immunotherapy is mainly attributed to poor tumor immunogenicity as well as the immunosuppressive tumor microenvironment(TME)leading to failure of immune response.Numerous therapeutic strategies including chemotherapy,radiotherapy,photodynamic,photothermal,magnetic,chemodynamic,sonodynamic and oncolytic therapy,have been developed to induce immunogenic cell death(ICD)of cancer cells and thereby elicit immunogenicity and boost the antitumor immune response.However,many challenges hamper the clinical application of ICD inducers resulting in modest immunogenic response.Here,we outline the current state of using nanomedicines for boosting ICD of cancer cells.Moreover,synergistic approaches used in combination with ICD inducing nanomedicines for remodeling the TME via targeting immune checkpoints,phagocytosis,macrophage polarization,tumor hypoxia,autophagy and stromal modulation to enhance immunogenicity of dying cancer cells were analyzed.We further highlight the emerging trends of using nanomaterials for triggering amplified ICD-mediated antitumor immune responses.Endoplasmic reticulum localized ICD,focused ultrasound hyperthermia,cell membrane camouflaged nanomedicines,amplified reactive oxygen species(ROS)generation,metallo-immunotherapy,ion modulators and engineered bacteria are among the most innovative approaches.Various challenges,merits and demerits of ICD inducer nanomedicines were also discussed with shedding light on the future role of this technology in improving the outcomes of cancer immunotherapy.

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.

Nanoengineered Shear-Thinning Hydrogel Barrier for Preventing Postoperative Abdominal Adhesions

More than 90%of surgical patients develop postoper-ative adhesions,and the incidence of hospital re-admissions can be as high as 20%.Current adhesion barriers present limited efficacy due to difficulties in application and incompatibility with minimally invasive interventions.To solve thisclinical limitation,we developed an injectable and sprayable shear-thinning hydrogel barrier(STHB)composed of silicate nanoplatelets and poly(ethylene oxide).We optimized this technology to recover mechanical integrity after stress,enabling its delivery though inject-able and sprayable methods.We also demonstrated limited cell adhesion and cytotoxicity to STHB compositions in vitro.The STHB was then tested in a rodent model of peritoneal injury to determine its e cacy preventing the formation of postoperative adhesions.After two weeks,the peritoneal adhesion index was used as a scoring method to determine the formation of postoperative adhesions,and STHB formulations presented superior e cacy compared to a commercially available adhesion barrier.Histological and immunohistochemical examination showed reduced adhesion formation and minimal immune infiltration in STHB formulations.Our technology demonstrated increased e cacy,ease of use in complex anatomies,and compatibility with di erent delivery methods,providing a robust universal platform to prevent postoperative adhesions in a wide range of surgical interventions.

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.

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.

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.

High-yield and rapid isolation of extracellular vesicles by flocculation via orbital acoustic trapping:FLOAT

Extracellular vesicles(EVs)have been identified as promising biomarkers for the noninvasive diagnosis of various diseases.However,challenges in separating EVs from soluble proteins have resulted in variable EV recovery rates and low purities.Here,we report a high-yield(>90%)and rapid(<10 min)EV isolation method called FLocculation via Orbital Acoustic Trapping(FLOAT).The FLOAT approach utilizes an acoustofluidic droplet centrifuge to rotate and controllably heat liquid droplets.By adding a thermoresponsive polymer flocculant,nanoparticles as small as 20 nm can be rapidly and selectively concentrated at the center of the droplet.We demonstrate the ability of FLOAT to separate urinary EVs from the highly abundant Tamm-Horsfall protein,addressing a significant obstacle in the development of EV-based liquid biopsies.Due to its high-yield nature,FLOAT reduces biofluid starting volume requirements by a factor of 100(from 20 mL to 200µL),demonstrating its promising potential in point-of-care diagnostics.

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.

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.

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.