This Review examines the transformative role of artificial intelligence (AI) in 3D bioprinting, focusing on how advanced AI technologies enhance its precision, functionality, and scalability. AI, through branches, such as machine learning (ML), computer vision (CV), robotics, natural language processing and expert systems, provides critical improvements in real-time process monitoring, error correction, and optimization of bioprinting parameters. The integration of AI enables automated quality control and predictive maintenance, improving bioprinting outcomes by increasing cell viability and structural fidelity, and reducing the amount of bioink wasted. Specifically, ML algorithms are employed to predict optimal bioprinting conditions and streamline the bioprinting workflow, while deep learning enhances the ability to process complex datasets for precision tissue biofabrication. Furthermore, AI-powered robotics and CV systems ensure accurate bioink placement and facilitate the construction of complex tissues. Despite the remarkable progress, challenges remain, particularly in the areas of process monitoring, quality control, and the scalability of bioprinting systems. This Review also aims to guide scientists, engineers, and healthcare providers in understanding the complexities and potential of AI-enhanced bioprinting, fostering a deeper appreciation of its role in the future of regenerative medicine and personalized healthcare.

Surface modification of nanoparticles (NPs) via the layer-by-layer (LbL) technique is a promising approach to generate targeted drug delivery vehicles. LbL-NPs have been successfully used in preclinical models for controlled drug release, tumor and immune cell targeting, improved pharmacokinetics and biodistribution, and controlling cellular trafficking and uptake mechanisms. A simple and scalable synthesis method for LbL-NPs that can be adapted for clinical translation is of great interest. Here we present a new method of polymer deposition onto NPs enabled through microfluidic (MCF) mixing. NPs are mixed with polyelectrolytes using commercially available bifurcating mixer MCF cartridges. In addition to increased process robustness, MCF allows for LbL electrostatic assembly using titrated polymer-to-NP weight equivalent ratios where no excess polymer is required to achieve a given LbL layering. Under such conditions, no time-consuming purification is needed, greatly increasing LbL-NP throughput and avoiding the loss of NPs during purification. We demonstrate the utility of this system using IL-12-loaded liposomal NPs which show equivalent efficacy in vitro and in vivo to LbL-NPs generated via traditional lab-scale batch-wise polymer adsorption and tangential flow filtration purification. Moreover, we show that MCF can assemble LbL films of various chemistries and on various NP core substrates.

Tissue development and regeneration are governed by processes that span subcellular signaling, cell-cell interactions, and the integrated mechanical properties of cellular collectives with their extracellular matrix. Synthetic biomaterials that can emulate the hierarchical structure and supracellular mechanics of living systems are paramount to the realization of regenerative medicine. Recent reports detail directed cell alignment on mechanically anisotropic but stiff liquid crystalline polymer networks (LCNs). While compelling, the potential implementation of these materials as tissue engineering scaffolds may be hindered by the orders of magnitude larger stiffness than most soft tissue. Accordingly, this report prepares liquid crystalline hydrogels (LCHs) that synergize the anisotropic mechanical properties intrinsic to LCNs with the cytocompatibility and soft mechanics of PEG hydrogels. LCH are prepared via sequential oligomerization and photopolymerization reactions between liquid crystalline (LC) monomers and poly(ethylene glycol) (PEG)-dithiol. Despite their low liquid crystalline content, swollen LCH oligomers are amenable to rheological alignment via direct ink write 3D printing. Mechanically anisotropic LCHs support C2C12 myoblast culture on their surface and direct their alignment in the stiffest direction. Further, C2C12s can be encapsulated within LCH oligomers and 3D-printed, whereby mechanical anisotropy of the LCH directs myoblast polarization in 3D.

Injecting nerve-blocking agents near peripheral nerves is a clinical option for treating post-operative and chronic pain. However, the peripheral nerve barriers (PNBs) pose a physiological barrier that hinders the permeation of nerve-blocking agents through PNBs, ultimately affecting their efficacy on neurons. Here, glucose-modified nanomicelles (Glu-NMs) are developed that are self-assembled from glucose-functionalized 3-aminophenylboronic acid-polyethylene glycol- stearic acid polymer (GLU-PBA-PEG-SA) and Pluronic P. It is demonstrated that Glu-NMs exhibit enhanced cellular uptake by perineurial cells and improved permeation through PNBs in rats, compared to unmodified nanomicelles (NMs). This enhancement is facilitated by carrier-mediated transport through glucose transporters. A single injection of capsaicin-loaded Glu-NMs (CAP@Glu-NMs) containing 1 mg of capsaicin at the sciatic nerves of rats resulted in nociceptive-selective nerve blockade lasting for 48.0 ± 19.6 h, which is seven times longer than the duration achieved with an equivalent dose of plain capsaicin and twice as long as that of capsaicin-loaded NMs (CAP@NMs). Furthermore, the CAP@Glu-NMs significantly mitigate capsaicin-associated side effects and result in benign local tissue reactions.

The therapeutic potential of RNA and DNA is evident from numerous formulations approved by the FDA in recent years, with formulations based on RNAi standing out as a successful example. The new class of medicines based on RNAi combines the process of diagnosis and treatment via sequence-specific recognition of biomarker mRNAs and downregulation of their translation. While this approach proved clinically successful, safer, and more personalized options that mitigate adverse effects can be revealed by separating diagnostic and treatment steps. A concept is introduced that allows RNAi therapies to selectively exert their function within diseased cells. The reconfigurable nucleic acid nanoparticles, or recNANPs, recognize overexpressed cancer biomarkers and conditionally release RNAi inducers targeting apoptosis inhibitors in pancreatic cancer cells. RecNANPs are non-immunostimulatory, achieve prolonged gene silencing compared to conventional RNAi inducers, and can be combined with chemotherapy. It is anticipated that this modular platform will enable further advancements in the development of biocompatible nanodevices activated by intracellular variables of choice, facilitating treatment with a repertoire of nucleic acid therapies.

Excessive inflammatory responses following traumatic brain injury (TBI) hinder tissue healing and impair long-term functional recovery. In addition to therapeutic strategies that directly target the core pathological mechanisms of TBI, biomaterials and regenerative medicine now offer promising new avenues. Here, we report a new formulation of an injectable hydrogel based on gelatin methacryloyl (GelMA) conjugated with β-cyclodextrin (βCD), designed for implantation in TBI lesions. Compared to GelMA alone, βCD-containing hydrogels (βCD-GelMA) demonstrated a strong capacity to sequester cholesterol from cultured cells, thereby reducing lipid droplet formation and suppressing inflammatory responses. Notably, aberrant cholesterol accumulation and lipid droplet formation were observed in a mouse model of TBI, andimplantation of βCD-GelMA into the lesion area significantly reduced peak tissue cholesterol and lipid droplet levels post-injury. More importantly, βCD-GelMA mitigated gliosis, reduced inflammation and scar formation, and improved functional outcomes in the TBI model. Mechanistically, by scavenging cholesterol from injured tissue, βCD-GelMA normalized microglial lipid droplet and promoted polarization toward a less inflammatory phenotype. In summary, our study highlights the strong therapeutic potential of βCD in restoring cholesterol homeostasis and resolving lipid droplet-associated inflammation, and demonstrates that the βCD-GelMA functional hydrogel represents a promising intervention strategy for head injuries.

The initiation of endometriotic lesions is not well understood or characterized because endometriosis is typically diagnosed at an advanced stage. Endometriotic lesions are most often found on pelvic tissues and organs, especially the ovaries. To investigate the role of tissue tropism on ovarian endometrioma initiation, we adapted a well-characterized polyacrylamide microarray system to investigate the role of tissue-specific extracellular matrix and adhesion motifs on endometriotic cell attachment, morphology, and size. We report the influence of cell origin (endometriotic vs. non-endometriotic), substrate stiffness mimicking aging and fibrosis, and the role of multicellular (epithelial-stromal) cohorts on cell attachment patterns. We identify multiple ovarian-specific attachment motifs that significantly increase endometriotic (vs. non-endometriotic) cell cohort attachment that could be implicated in early disease etiology.

The increasing risks of ionizing radiation from deep space travel and nuclear accidents necessitate the development of effective countermeasures. Acute radiation syndrome (ARS), particularly hematopoietic ARS (H-ARS), leads to life-threatening anemia and bone marrow failure, with long-term risks including cancer and cardiovascular disease. This study presents phenylboronic acid-functionalized chitosan-polyethylenimine (CPB) polymers designed for efficient oral delivery of pegfilgrastim (PF), an FDA-approved radioprotective agent. Nanoparticles were prepared through complexation of PF with tannic acid, forming a negatively charged core, followed by encapsulation with a CPB polymer shell. This supramolecular strategy enabled efficient protein condensation into uniform nanoparticles. The nanosystem effectively reduced H-ARS-associated anemia in mice by promoting blood cell reconstitution, supported by results with human hematopoietic stem/progenitor cells. Additionally, the material reduced toll-like receptor activation in multiple human cell types post-radiation. This system may mitigate radiation injury risks from accidental exposure on Earth and during extended space missions.

Hydrogels are routinely used as scaffolds to mimic the extracellular matrix for tissue engineering. However, common strategies to covalently crosslink hydrogels employ reaction conditions with potential off-target biological reactivity. The limited number of suitable bioorthogonal chemistries for hydrogel crosslinking restricts how many material properties can be independently addressed to control cell fate. To expand the bioorthogonal toolkit available for hydrogel crosslinking, we identify isonitrile ligations as a promising class of reactions. Isonitriles are compact, stable, selective, and biocompatible moieties that react with chlorooxime (ChO), tetrazine (Tz), and azomethine imine (AMI) functional groups under physiological conditions. We demonstrate that all three ligation reactions can form hydrogels, with isonitrile-ChO ligation exhibiting optimal gelation properties. Synthetic poly(ethylene glycol) (PEG) hydrogels crosslinked by isonitrile-ChO ligation exhibit rapid gelation kinetics, elastic mechanical properties, stability under physiological conditions, and high biocompatibility. By combining ChO-functionalized multi-arm PEGs with isonitrile-functionalized engineered elastin-like proteins (ELPs), we demonstrate simultaneous control over network connectivity and adhesive ligand presentation, which in turn regulate cell spreading. These hydrogels enable the long-term culture of numerous human cell types relevant to regenerative medicine. Furthermore, we demonstrate that isonitrile-ChO ligation is orthogonal to common azide-alkyne cycloaddition, enabling independent, bioorthogonal functionalization of hydrogels containing live cells.

The field of cancer biology and therapeutics has soared in the past several decades with new therapeutic modalities and options for patients, such as chemoradiotherapy, immunotherapy, and combination therapy. This dramatic success in expanding patient options is primarily attributed to the development of various model systems to elucidate drivers of oncogenesis, tumor maturation and evolution, and response to therapeutics. While mouse models have been a workhorse of cancer research, technological progress in patient-derived tumor models has afforded more tunable and scrutable systems for patient-predictive platforms and mechanistic study. This review explores the technological innovations in 3D solid tumor models and their applicability to various aspects of cancer biology and identification of therapeutics. Features of the tumor and tumor microenvironment like spatial heterogeneity, multicellular populations and genomic variations are addressed and elaborated through the establishment of new models. We further address the integration of perfusable vasculature with 3D tumor models and the potentially wide-ranging applications of these more complex platforms in precision medicine and cancer immunotherapy. Finally, we provide an outlook on the future of experimental cancer models for both biological investigation and bench-to-bedside pipeline development.

Drug-resistant microorganisms cause serious problems in human healthcare, leading to the persistence in infections and poor treatment outcome from conventional therapy. In this study, we introduce a gene targeting strategy using microbubble-controlled nanoparticles that can effectively eliminate biofilms of methicillin-resistant (MRSA) . We developed biofilm-targeting nanoparticles (BTN) capable of delivering oligonucleotides that effectively remove biofilm-associated bacteria upon controlled delivery with diatom-based microbubblers (MB). We validated the activity of BTN in silencing key bacterial genes related to MRSA biofilm formation (), bacterial growth (), and antimicrobial resistance (), as well as their multi-targeting ability . We next examined the integration of BTN with MB, resulting in synergistic effects in biofilm removal and antimicrobial activity in an porcine skin model. We further investigated the therapeutic efficacy in a mouse wound model infected with MRSA biofilm, which showed that MB-controlled BTN delivery substantially reduced bacterial load and led to the effective elimination of the biofilm. This study underscores the potential of the gene silencing platform with physical enhancement as a promising strategy to combat problems related to biofilms and antibiotic resistance.

Epidural electrical stimulation (EES) of the spinal cord is widely applied for pain management and has garnered considerable interest as a possible route to functional restoration after spinal cord injury. Currently, EES employs bulky, non-conformal paddle arrays with low channel counts. This limits stimulation effectiveness by requiring high stimulation currents, reduces selectivity of muscle recruitment, and requires subject-specific designs to accommodate varied neuroanatomy across the patient population. Here, we report on a thin-film, high-channel count microelectrode array, termed SpineWrap, which wraps around the dorsolateral aspect of the rat spinal cord. SpineWrap delivers focal stimulation to selectively activate muscles due to its unique design features, including its thin substrate, high conformability, high channel count, on-device ground, and the material properties of its platinum nanorod contacts. Through computational and in vivo studies, we show that SpineWrap can selectively recruit muscles in the rat lower limb and identify stimulation hotspots at a submillimeter resolution, maximizing muscle recruitment selectivity. We also investigate the effect of channel count and density on muscle recruitment selectivity and show that rat spinal cord arrays require submillimeter pitches to achieve maximal selectivity. SpineWrap represents an advancement in EES technology and, when adapted to be used chronically, has the potential to improve SCI treatment by providing more refined stimulation.

Aggressive cancers, characterized by high metastatic potential and resistance to conventional therapies, present a significant challenge in oncology. Current treatments often fail to effectively target metastasis, recurrence, and the immunosuppressive tumor microenvironment, while causing significant off-target toxicity. Here, we present superparamagnetic copper iron oxide nanoparticles (SCIONs) as a multifunctional platform that integrates magnetic hyperthermia therapy, immune modulation, and targeted chemotherapeutic delivery, aiming to provide a more comprehensive cancer treatment. Specifically, SCIONs generate localized hyperthermia under an alternating magnetic field while delivering a copper-based anticancer agent, resulting in a synergistic anticancer effect. The hyperthermia induced by SCIONs caused ER stress and ROS production, leading to significant tumor cell death, while the copper complex further enhanced oxidative stress, ferroptosis, and apoptosis. Beyond direct cytotoxicity, SCIONs disrupted the tumor microenvironment by inhibiting cancer-associated fibroblasts, downregulating epithelial-mesenchymal transition markers, and reducing cell migration and invasion, thereby limiting metastasis. Additionally, SCION-based therapy reprogrammed the immune microenvironment by inducing immunogenic cell death and enhancing dendritic cell activation, resulting in increased CD8+ T cell infiltration and amplified antitumor immunity. This integrated approach targets primary and metastatic tumors while mitigating immunosuppression, offering a promising next-generation therapy for combating cancer with enhanced efficacy and reduced side effects.

Despite notable advancements, the significantly improved yet suboptimal heating efficiency of current magnetic nanoparticles hinders the effectiveness of systemically delivered magnetic hyperthermia in reducing tumor size or halting growth. Addressing this challenge, the seed-and-growth thermal decomposition method has been developed to synthesize cobalt-doped iron oxide nanoparticles featuring a cubical bipyramid morphology and consisting of both magnetite and maghemite phases within their nanostructure. They possess an exceptional specific absorption rate of 14,686 ± 396 W g Fe, inducing a temperature rise of 3.73°C per second when subjected to an alternating magnetic field (315 kHz; 26.8 kA m). The cubical bipyramid-shaped nanoparticles, functionalized with a cancer-targeting LHRH peptide, efficiently accumulate in ovarian cancer xenografts following an intravenous injection at a relatively low dose of 4 mg kg, elevating intratumoral temperatures beyond 50°C with a highly efficient heating rate. In contrast to previously reported magnetic nanoparticles with ultrahigh heating efficiency, the developed cubical bipyramid-shaped nanoparticles effectively halt ovarian cancer tumor growth after a single 30-minute session of magnetic hyperthermia. These outcomes underscore the profound potential of shape-dependent magnetic hyperthermia, where the unique cubical bipyramid morphology significantly enhances the heating efficiency and therapeutic efficacy of magnetic nanoparticles, revolutionizing the design of magnetic nanomaterials and significantly improving the effectiveness of hyperthermia-based cancer treatments.

Non-invasive imaging modalities that identify rupture-prone atherosclerotic plaques hold promise to improve patient risk stratification and advance early intervention strategies. Here, phase-changing peptide nanoemulsions are developed as theranostic contrast agents for synchronous ultrasound detection and therapy of at-risk atherosclerotic lesions. By targeting lipids within atherogenic foam cells, and exploiting characteristic features of vulnerable plaques, these nanoemulsions preferentially accumulate within lesions and are retained by intraplaque macrophages. It is demonstrated that acoustic vaporization of intracellular nanoemulsions promotes lipid efflux from foam cells and generates echogenic microbubbles that provide contrast-enhanced ultrasound identification of lipid-rich anatomical sites. In Doppler mode, stably oscillating peptide nanoemulsions induce random amplitude and phase changes of the echo wave to generate transient color imaging features, referred to as 'twinkling'. Importantly, acoustic twinkling is unique to these peptide emulsions, and not observed from endogenous tissue bubble nuclei, generating diagnostic features that offer unprecedented spatial precision of lesion identification in 3D.

Cell reprogramming and manufacturing have broad applications in tissue regeneration and disease treatment. However, many derived cell types lack unique cell surface markers for protein-based cell sorting, making it difficult to isolate these cells from mixed populations. Additionally, there is a need to identify and isolate cells of interest at the early stages of cell expansion. To address this challenge, we engineered a nucleic acid-based gold nanorod (NAGNR) fluorescent biosensor that can detect the mRNA expression of intracellular markers for cell sorting. We demonstrated its application in isolating induced neuronal (iN) cells from dermal fibroblast populations during the early stages of cell reprogramming. Cell sorting based on the mRNA of the neuronal transcriptional factor Ascl1 resulted in an enrichment of iN cells from 3% to 72%, and additional sorting with the transcriptional factor Scn2 further increased iN enrichment. Moreover, NAGNR biosensors can be used in conjunction with protein marker-based cell sorting. NAGNR-sorted iN cells show a functional response to electrical stimulation in a co-culture of iN cells and muscle cells. These findings demonstrate that NAGNR-based cell sorting offers great potential for cell identification and isolation at an early stage of cell reprogramming and manufacturing.

The ability to precisely arrange and control the assembly of diverse cell types into intricate three-dimensional (3D) structures remains a critical challenge in tissue engineering. Herein, we describe a versatile and programmable 3D cell sheet assembly technology by developing biodegradable nanochannel (BNC) membrane to fulfill this unmet need. This membrane, hierarchically assembled from two-dimensional nanomaterial aggregates, exhibits both exceptional fluid permeability and rapid biodegradation under physiological conditions. The unique properties of the BNC membrane enable precise spatial and temporal control over cell assembly, facilitating the creation of complex 3D cellular architectures. The BNC membrane was integrated with a programmable negative-pressure-based cell assembly strategy to form single and multi-cellular 3D sheets in a highly controllable manner. To demonstrate the feasibility and translatability of this technology in the field of tissue engineering, we devised approaches to screen stem cell-derived therapeutics with "core-shell" macrophage-fibroblast multi-cellular patterns and treat murine diabetic skin wounds via scaffold-free 3D adipose-derived mesenchymal stem cell (ADMSC) sheets. In summary, our results demonstrate that the BNC membrane-based 3D cell sheet assembly approach significantly advances current tissue engineering capabilities, offering substantial potential for both regenerative medicine applications and the development of physiologically relevant disease models.

Injectable hydrogels represent a promising strategy for the extended release of biological molecules, thereby reducing the frequency of injections. This study introduces a novel system based on Michael addition of dextran and polyethylene glycol (PEG) polymers functionalized with oxanorbornadiene (OND) and thiol groups, respectively. Reliable control over gelation speed allows administration by injection using a simple syringe-to-syringe mixing protocol that entrains more than 95% of virus-like particle (VLP) cargo. A combination of retro-Diels-Alder and hydrolytic ester bond cleavage gives rise to programmable release of the VLPs. Different release profiles, including burst, linear, and delayed release over a two-week period, are engineered using different OND linkages, and rheological characterization shows the hydrogels to be well within the desired range of stiffness for subcutaneous use. The modular nature of this system offers a generalizable platform for developing degradable materials aimed at sustained release biomedical applications.

Enteric neurons are critical in maintaining organ homeostasis within the small intestine, and their dysregulation are implicated in gastrointestinal disorders and neurodegenerative diseases. Most in vitro models lack enteric innervation, limiting basic discovery and disease modeling research. Here, a high-throughput 3D microphysiological system (MPS), or organ chip is presented that supports a primary epithelial monolayer interfacing directly with encapsulated primary enteric neurons. The device features twelve 3D MPSs per device and gravity-driven flow via a laboratory rocker to induce biomimetic shear stress on the epithelium culture and provide continuous nutrient presentation. Intestinal and neural tissue exhibited expected morphologies. Neural gene upregulation in the epithelium suggests RNA contamination from proximal enteric neurons extending neurites toward the epithelial monolayer. With the enteric nervous system (ENS), barrier integrity significantly increased for both TEER and permeability assays, a 1.25-fold greater resistance and 10% lower permeability as compared to epithelium cultured alone. The presence of the ENS resulted in a significant (1.4-fold) reduction in epidermal growth factor (EGF). Additionally, several key epithelial genes are compared between duodenal tissue and epithelial monolayers with and without neurons present. Results demonstrated changes in cytokine gene expression and WNT pathways, highlighting innervation is essential to create more biomimetic and physiologically relevant in vitro models.

Neuromodulation technologies have gained considerable attention for their clinical potential in treating neurological disorders and advancing cognition research. However, traditional methods like electrical stimulation and optogenetics face technical and biological challenges that limit their therapeutic and research applications. A promising alternative, photoelectric neurostimulation, uses near-infrared light to generate electrical pulses and thus enables stimulation of neuronal activity without genetic alterations. This study explores various design strategies to enhance photoelectric stimulation with minimally invasive, ultrasmall, untethered carbon electrodes. Employing a multiphoton laser as the near-infrared (NIR) light source, benchtop experiments are conducted using a three-electrode setup and chronopotentiometry to record photo-stimulated voltage. In vivo evaluations utilize Thy1-GCaMP6s mice with acutely implanted ultrasmall carbon electrodes. Results highlighted the beneficial effects of high duty-cycle laser scanning and photovoltaic polymer interfaces on the photo-stimulated voltages by the implanted electrode. Additionally, the promising potential of carbon-based diamond electrodes are demonstrated for photoelectric stimulation and the application of photoelectric stimulation in precise chemical delivery by loading mesoporous silica nanoparticles (SNPs) co-deposited with polyethylenedioxythiophene (PEDOT). Together, these findings on photoelectric stimulation utilizing ultrasmall carbon electrodes underscore its immense potential for advancing the next generation of neurostimulation technology.

The biophysical heterogeneity of the bone-cartilage interface requires complex materials to mimic differences in bone density, extracellular matrix composition, and mineralization. Biomaterial approaches to repair osteochondral tissue typically use multilayer scaffolds, which require multi-step fabrication and may undergo delamination at the construct interface. This work describes the development of functionalized microgels for the repair of osteochondral tissues using an N-cadherin peptide, BMP-2 peptide, and changes in stiffness to create pro-osteogenic and pro-chondrogenic microgels. Microgels, when annealed into a scaffold, outperformed bulk hydrogel controls evidenced by upregulation of osteogenic and chondrogenic markers in mesenchymal stromal cells (MSCs). The macroporous void space present in microgel annealed scaffolds enabled robust cell proliferation and ECM deposition throughout the entire scaffold. We then created a bilayer functionalized annealed microgel scaffold and assessed the ability to spatially control the differentiation of MSCs. Osteochondral bilayer scaffolds exhibited distinct regions of osteogenic and chondrogenic protein expression as a function of microgel population upon immunostaining for osteocalcin and aggrecan, respectively. Spatial transcriptomics confirmed osteogenic and chondrogenic genes were upregulated in their respective microgel regions. These studies highlight the tunable and functionalizable nature of microgels and the importance of macroporous void space.