Tissue interface restoration poses significant challenges in tissue engineering, particularly in areas requiring gradients of cellularity, biochemical composition, and mechanical properties essential for tissue-specific functions. Recent advancements in microfluidic technology have enabled the creation of hydrogels with spatially defined gradients of biological molecules for engineering gradient tissues that mimic the natural heterogeneity of the native extracellular matrix. However, these gradients are typically outside relevant millimeter length scales for biological interfaces. To address these challenges, a branched microfluidic system capable of generating millimeter-scale hydrogels with dual-opposing gradients of RNAi molecules nanocomplexed with thiolated polyethyleneimine is demonstrated. These nanocomplexes are incorporated into photocrosslinkable poly(ethylene glycol)-diacrylate (PEG-DA) monomer solutions, which are then injected and mixed within a PDMS microfluidic chip featuring a branched channel network. The process results in stable, linear, 3-mm dual-opposing RNA gradients within the hydrogel, which is subsequently photocrosslinked. The generated hydrogels demonstrate precise spatial regulation of gene expression within encapsulated cells, as confirmed by fluorescence analysis. This platform holds significant potential for engineering complex tissue constructs and enabling the targeted delivery of RNAi molecules, influencing both encapsulated and endogenous cells. This advancement could play a crucial role in the regeneration of critical tissue interfaces such as tendon-to-bone and cartilage-to-bone.

This study introduces a hyaluronic acid hydrogel-based microneedle patch embedded with IL-4-loaded PLGA microparticles. This highly biodegradable cardiac patch is for localized immunomodulation and cardiac repair following acute myocardial infarction (MI). This patch enables sustained IL-4 release, thereby fostering a macrophage-mediated pro-reparative microenvironment that promotes cardiomyocyte re-entry into the cell cycle and attenuates endothelial inflammatory signaling. In both rat and porcine MI models, this treatment significantly improved cardiac function and preserved myocardial structure. These findings highlight the translational potential of this patch as a therapeutic strategy for heart repair during the acute phase of MI.

Extracellular matrices of living tissues exhibit viscoelastic properties, yet how these properties regulate chromatin and the epigenome remains unclear. Here, we show that viscoelastic substrates induce changes in nuclear architecture and epigenome, with more pronounced effects on softer surfaces. Fibroblasts on viscoelastic substrates display larger nuclei, lower chromatin compaction, and differential expression of distinct sets of genes related to the cytoskeleton and nuclear function, compared to those on elastic surfaces. Slow-relaxing viscoelastic substrates reduce lamin A/C expression and enhance nuclear remodeling. These structural changes are accompanied by a global increase in euchromatin marks and local increase in chromatin accessibility at cis-regulatory elements associated with neuronal and pluripotent genes. Consequently, viscoelastic substrates improve the reprogramming efficiency from fibroblasts into neurons and induced pluripotent stem cells. Collectively, our findings unravel the roles of matrix viscoelasticity in epigenetic regulation and cell reprogramming, with implications for designing smart materials for cell fate engineering.

The field of mechanobiology is gaining prominence due to recent findings that show cells sense and respond to the mechanical properties of their environment through a process called mechanotransduction. The mechanical properties of cells, cell organelles, and the extracellular matrix are understood to be viscoelastic. Various technologies have been researched and developed for measuring the viscoelasticity of biological materials, which may provide insight into both the cellular mechanisms and the biological functions of mechanotransduction. Here, we explain the concept of viscoelasticity and introduce the major techniques that have been used to measure the viscoelasticity of various soft materials in different length- and timescale frames. The topology of the material undergoing testing, the geometry of the probe, the magnitude of the exerted stress, and the resulting deformation should be carefully considered to choose a proper technique for each application. Lastly, we discuss several applications of viscoelasticity in 3D cell culture and tissue models for regenerative medicine, including organoids, organ-on-a-chip systems, engineered tissue constructs, and tunable viscoelastic hydrogels for 3D bioprinting and cell-based therapies.

Synthetic Notch (synNotch) receptors are modular synthetic components that are genetically engineered into mammalian cells to detect signals presented by neighboring cells and respond by activating prescribed transcriptional programs. To date, synNotch has been used to program therapeutic cells and pattern morphogenesis in multicellular systems. However, cell-presented ligands have limited versatility for applications that require spatial precision, such as tissue engineering. To address this, we developed a suite of materials to activate synNotch receptors and serve as generalizable platforms for generating user-defined material-to-cell signaling pathways. First, we demonstrate that synNotch ligands, such as GFP, can be conjugated to cell- generated ECM proteins via genetic engineering of fibronectin produced by fibroblasts. We then used enzymatic or click chemistry to covalently link synNotch ligands to gelatin polymers to activate synNotch receptors in cells grown on or within a hydrogel. To achieve microscale control over synNotch activation in cell monolayers, we microcontact printed synNotch ligands onto a surface. We also patterned tissues comprising cells with up to three distinct phenotypes by engineering cells with two distinct synthetic pathways and culturing them on surfaces microfluidically patterned with two synNotch ligands. We showcase this technology by co-transdifferentiating fibroblasts into skeletal muscle or endothelial cell precursors in user-defined spatial patterns towards the engineering of muscle tissue with prescribed vascular networks. Collectively, this suite of approaches extends the synNotch toolkit and provides novel avenues for spatially controlling cellular phenotypes in mammalian multicellular systems, with many broad applications in developmental biology, synthetic morphogenesis, human tissue modeling, and regenerative medicine.

The use of synthetic antigen-presenting cells to activate and expand engineered T cells for the treatment of cancers typically results in therapies that are suboptimal in effectiveness and durability. Here we describe a high-throughput microfluidic system for the fabrication of synthetic cells mimicking the viscoelastic and T-cell-activation properties of antigen-presenting cells. Compared with rigid or elastic microspheres, the synthetic viscoelastic T-cell-activating cells (SynVACs) led to substantial enhancements in the expansion of human CD8+ T cells and to the suppression of the formation of regulatory T cells. Notably, activating and expanding chimaeric antigen receptor (CAR) T cells with SynVACs led to a CAR-transduction efficiency of approximately 90% and to substantial increases in T memory stem cells. The engineered CAR T cells eliminated tumour cells in a mouse model of human lymphoma, suppressed tumour growth in mice with human ovarian cancer xenografts, persisted for longer periods and reduced tumour-recurrence risk. Our findings underscore the crucial roles of viscoelasticity in T-cell engineering and highlight the utility of SynVACs in cancer therapy. Synthetic cells mimicking the viscoelastic and T-cell-activation properties of antigen-presenting cells provide substantial enhancements in the expansion and potency of engineered human cytotoxic T cells.

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, a nucleic acid-based gold nanorod (NAGNR) fluorescent biosensor was engineered to detect the mRNA expression of intracellular markers for cell sorting. Its application is demonstrated 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.

We investigate how matrix stiffness regulates chromatin reorganization and cell reprogramming and find that matrix stiffness acts as a biphasic regulator of epigenetic state and fibroblast-to-neuron conversion efficiency, maximized at an intermediate stiffness of 20 kPa. ATAC sequencing analysis shows the same trend of chromatin accessibility to neuronal genes at these stiffness levels. Concurrently, we observe peak levels of histone acetylation and histone acetyltransferase (HAT) activity in the nucleus on 20 kPa matrices, and inhibiting HAT activity abolishes matrix stiffness effects. G-actin and cofilin, the cotransporters shuttling HAT into the nucleus, rises with decreasing matrix stiffness; however, reduced importin-9 on soft matrices limits nuclear transport. These two factors result in a biphasic regulation of HAT transport into nucleus, which is directly demonstrated on matrices with dynamically tunable stiffness. Our findings unravel a mechanism of the mechano-epigenetic regulation that is valuable for cell engineering in disease modeling and regenerative medicine applications.

The role of transcription factors and biomolecules in cell type conversion has been widely studied. Yet, it remains unclear whether and how intracellular mechanotransduction through focal adhesions (FAs) and the cytoskeleton regulates the epigenetic state and cell reprogramming. Here, it is shown that cytoskeletal structures and the mechanical properties of cells are modulated during the early phase of induced neuronal (iN) reprogramming, with an increase in actin cytoskeleton assembly induced by Ascl1 transgene. The reduction of actin cytoskeletal tension or cell adhesion at the early phase of reprogramming suppresses the expression of mesenchymal genes, promotes a more open chromatin structure, and significantly enhances the efficiency of iN conversion. Specifically, reduction of intracellular tension or cell adhesion not only modulates global epigenetic marks, but also decreases DNA methylation and heterochromatin marks and increases euchromatin marks at the promoter of neuronal genes, thus enhancing the accessibility for gene activation. Finally, micro- and nano-topographic surfaces that reduce cell adhesions enhance iN reprogramming. These novel findings suggest that the actin cytoskeleton and FAs play an important role in epigenetic regulation for cell fate determination, which may lead to novel engineering approaches for cell reprogramming.

Abstract Immunotherapy shows great promise for cancer treatment, but its effectiveness and durability remain limited. Thus, there is unmet needs for more robust technology for immune cell engineering. Here we develop a scalable microfluidic platform to fabricate synthetic viscoelastic activating cells (SynVACs) with programmable mechanical and chemical properties. We demonstrate that the viscoelastic nature of SynVACs significantly impacts T cell functionality. Compared to rigid or elastic microspheres, SynVACs greatly enhance human T cell expansion with drastic CD8+ T cell generation while suppressing regulatory T cell formation, resulting in enhanced tumor killing capability. Notably, expanding chimeric antigen receptor (CAR)-T cells with SynVACs achieves approximately 90% CAR transduction efficiency and leads to a six-fold increase of T memory stem cells. These engineered CAR-T cells exhibit superior efficacy in eliminating tumor cells, not only in a human lymphoma mouse model, but also in a solid tumor xenograft mouse model of ovarian cancer. Additionally, SynVAC-expanded CAR-T cells persist for longer time periods in vivo to suppress tumor growth and recurrence. These findings underscore the crucial role of mechanical signals in T cell engineering and highlight the potential of SynVAC platform in CAR-T therapy and broad immunoengineering applications.

Cell reprogramming has wide applications in tissue regeneration, disease modelling and personalized medicine. In addition to biochemical cues, mechanical forces also contribute to the modulation of the epigenetic state and a variety of cell functions through distinct mechanisms that are not fully understood. Here we show that millisecond deformation of the cell nucleus caused by confinement into microfluidic channels results in wrinkling and transient disassembly of the nuclear lamina, local detachment of lamina-associated domains in chromatin and a decrease of histone methylation (histone H3 lysine 9 trimethylation) and DNA methylation. These global changes in chromatin at the early stage of cell reprogramming boost the conversion of fibroblasts into neurons and can be partially reproduced by inhibition of histone H3 lysine 9 and DNA methylation. This mechanopriming approach also triggers macrophage reprogramming into neurons and fibroblast conversion into induced pluripotent stem cells, being thus a promising mechanically based epigenetic state modulation method for cell engineering. Mechanical confinement of fibroblasts into micrometre-sized channels deforms the cell nucleus, leading to temporary nuclear lamina destablization and disassembly, loss of lamina-associated domains in chromatin and a decrease in histone and DNA methylation. These mechanically induced alterations in chromatin boost the conversion of fibroblasts into neurons and pluripotent stem cells and thus can be explored for cell engineering applications.

Silk fibroin (SF) is a promising biomaterial for tendon repair, but its relatively rigid mechanical properties and low cell affinity have limited its application in regenerative medicine. Meanwhile, gelatin-based polymers have advantages in cell attachment and tissue remodeling but have insufficient mechanical strength to regenerate tough tissue such as tendons. Taking these aspects into account, in this study, gelatin methacryloyl (GelMA) is combined with SF to create a mechanically strong and bioactive nanofibrous scaffold (SG). The mechanical properties of SG nanofibers can be flexibly modulated by varying the ratio of SF and GelMA. Compared to SF nanofibers, mesenchymal stem cells (MSCs) seeded on SG fibers with optimal composition (SG7) exhibit enhanced growth, proliferation, vascular endothelial growth factor production, and tenogenic gene expression behavior. Conditioned media from MSCs cultured on SG7 scaffolds can greatly promote the migration and proliferation of tenocytes. Histological analysis and tenogenesis-related immunofluorescence staining indicate SG7 scaffolds demonstrate enhanced in vivo tendon tissue regeneration compared to other groups. Therefore, rational combinations of SF and GelMA hybrid nanofibers may help to improve therapeutic outcomes and address the challenges of tissue-engineered scaffolds for tendon regeneration.

Electrostatically actuated microplates with multilayer and material anisotropy properties, are widely employed in microelectromechanical systems. However, previous theories rarely consider the aforementioned properties simultaneously, hindering their widespread application. This paper proposes a general theoretical model for electrostatically actuated rectangular multilayer anisotropic microplates subjected to residual stress and hydrostatic pressure by combining the classical laminated thin plate theory, Galerkin method and a partial expansion approach for nonlinear electrostatic force. This model enables successful establishment of closed-form expressions for the main mechanical behaviors, e.g. the pull-in voltage, static deflection, and resonant frequency. Validation of these expressions, using finite element method simulations and experimental results, shows significant improvement in the analysis accuracy (15 times higher) compared to those theories neglecting the material anisotropy, as well as excellent applicability across a wide range of DC voltages and dimensions. Additionally, the influences of electrostatic softening effects and scale effects on the theories are also discussed.

There is a pressing need for effective therapeutics for coronavirus disease 2019 (COVID-19), the respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. The process of drug development is a costly and meticulously paced process, where progress is often hindered by the failure of initially promising leads. To aid this challenge, in vitro human microphysiological systems need to be refined and adapted for mechanistic studies and drug screening, thereby saving valuable time and resources during a pandemic crisis. The SARS-CoV-2 virus attacks the lung, an organ where the unique three-dimensional (3D) structure of its functional units is critical for proper respiratory function. The in vitro lung models essentially recapitulate the distinct tissue structure and the dynamic mechanical and biological interactions between different cell types. Current model systems include Transwell, organoid and organ-on-a-chip or microphysiological systems (MPSs). We review models that have direct relevance toward modeling the pathology of COVID-19, including the processes of inflammation, edema, coagulation, as well as lung immune function. We also consider the practical issues that may influence the design and fabrication of MPS. The role of lung MPS is addressed in the context of multi-organ models, and it is discussed how high-throughput screening and artificial intelligence can be integrated with lung MPS to accelerate drug development for COVID-19 and other infectious diseases.

Infection by SARS-CoV-2 virus often induces the dysregulation of immune responses, tissue damage, and blood clotting. Engineered biomaterials from the nano- to the macroscale can provide targeted drug delivery, controlled drug release, local immunomodulation, enhanced immunity, and other desirable functions to coordinate appropriate immune responses and to repair tissues. Based on the understanding of COVID-19 disease progression and immune responses to SARS-CoV-2, we discuss possible immunotherapeutic strategies and highlight biomaterial approaches from the perspectives of preventive immunization, therapeutic immunomodulation, and tissue healing and regeneration. Successful development of biomaterial platforms for immunization and immunomodulation will not only benefit COVID-19 patients, but also have broad applications for a variety of infectious diseases.

Despite considerable efforts in modeling liver disease in vitro, it remains difficult to recapitulate the pathogenesis of the advanced phases of non-alcoholic fatty liver disease (NAFLD) with inflammation and fibrosis. Here, a liver-on-a-chip platform with bioengineered multicellular liver microtissues is developed, composed of four major types of liver cells (hepatocytes, endothelial cells, Kupffer cells, and stellate cells) to implement a human hepatic fibrosis model driven by NAFLD: i) lipid accumulation in hepatocytes (steatosis), ii) neovascularization by endothelial cells, iii) inflammation by activated Kupffer cells (steatohepatitis), and iv) extracellular matrix deposition by activated stellate cells (fibrosis). In this model, the presence of stellate cells in the liver-on-a-chip model with fat supplementation showed elevated inflammatory responses and fibrosis marker up-regulation. Compared to transforming growth factor-beta-induced hepatic fibrosis models, this model includes the native pathological and chronological steps of NAFLD which shows i) higher fibrotic phenotypes, ii) increased expression of fibrosis markers, and iii) efficient drug transport and metabolism. Taken together, the proposed platform will enable a better understanding of the mechanisms underlying fibrosis progression in NAFLD as well as the identification of new drugs for the different stages of NAFLD.