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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Three-dimensional silk fibroin microsphere-nanofiber scaffolds for vascular tissue engineering

A significant limitation in the engineering of artificial small-diameter vascular scaffolds is that the number of endothelial cells(ECs)is not sufficient to generate a confluent coverage of the vascular scaffolds,so that the surfaces of vascular scaffolds form thrombus via platelet adhesion and aggregation.Thrombus decrease relies on three-dimensional(3D)scaffolds to mimic the natural extracellular matrix(ECM)as templates to regulate cell behavior and facilitate tissue maturation.Here,we developed 3D scaffolds consisting of silk fibroin(SF)nanofibers and homogeneous microspheres by electrospinning and microfluidics.The nanofibers with diameters ranging from 250 to 350 nm doped with microspheres(2–10μm)formed bridge-shaped structures.ECs were seeded and maintained on the 3D microsphere-nanofiber scaffolds with a mean fiber diameter of 300 nm.A 10%higher ratio of cell proliferation on 3D microsphere-nanofiber SF scaffolds was noted as compared to that on microporous and sponge-like SF scaffolds with small surface network fabricated by freeze-drying.Moreover,the gene transcript levels including CD146,VE-C and PECAM-1 were better preserved on 3D microsphere-nanofiber SF scaffolds than those on freeze-dried scaffolds.Thus,the developed 3D microsphere-nanofiber structure may have a myriad of applications in vascular tissue engineering scaffolds and cardiovascular devices.

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.