Advanced Healthcare Materials

In the US, 33 million musculoskeletal injuries have been reported per year, with 50% involving tendons and ligaments in both athletic and aging populations. Tendon repair often results in the formation of biomechanically inferior scar tissue rather than functional regeneration. Local cell therapy is garnering significant interest in tendon repair because it provides a targeted means to repopulate defects with potent therapeutic cells. Here, we developed a porcine tendon-derived thermoresponsive extracellular matrix hydrogel (TG) as an injectable, bio-instructive carrier for scleraxis-overexpressing induced mesenchymal stem cell derived tenocytes (iTenocytes), with the goal of improving cell retention and overall transplantation success. TG was compositionally distinct from purified collagen (PC, from rat tail type 1 collagen) and exhibited favorable material properties for a minimally invasive percutaneous strategy, including thermoresponsive gelation, shear-thinning injectability, retention at the injection site, and controlled biodegradation. In vitro, TG supported three-dimensional cell residence, promoted cell interconnectivity and redistribution at the matrix interface, and increased collagen type I release from iTenocytes compared to those embedded in PC. Transcriptomic and proteomic analyses further showed that TG enhanced programs associated with tenogenic maturation, extracellular matrix assembly, focal adhesion, mechano-transduction, and remodeling. In a rat Achilles tendon partial defect model, both optical imaging and histological analyses demonstrated retention of iTenocytes at the injection site. Additionally, confocal imaging demonstrated retention of iTenocytes within the defect site for up to 10 days. Together, these findings identify TG as a biofunctional injectable carrier that supports the tenogenic characteristics of iTenocytes and enhances their local persistence after transplantation.

For advanced biomedical research, complex, well-described human blood-brain barrier (BBB) models are crucial for studying central nervous system pathologies and brain targeting of nanotherapeutics. Our aim was to establish and characterize a complex lab-on-a-chip (LOC) system integrating a BBB model with neural organoids (NO). We optimized the conditions for BBB-NO models under static and dynamic circumstances. The combination of a human stem cell-derived BBB co-culture model with human midbrain organoids in a microfluidic LOC allowed the observation of BBB and neural tissue changes and the separate analysis of the barrier and brain units. The LOC design enabled phase contrast and fluorescent microscopy on the whole brain endothelial culture surface, barrier integrity and permeability measurements across the BBB model, and molecule passage into NOs. BBB-specific endothelial morphology, gene and protein expression, and good barrier integrity were demonstrated, corroborating the strength of the LOC engineering and cellular design. The transport of targeted nanoparticles across the BBB model followed by their entry to neural organoids validated the barrier integrity and transporter functionality of the dynamic integrated complex model. As a proof-of-concept experiment to confirm the translational value of the BBB-NO model integrated in the LOC device, we examined a clinically used hyperosmolar iodinated contrast agent, iopamidol, with confirmed neurological side effects. We corroborated that iopamidol induces transient BBB dysfunction and neural effects in the complex system, not only validating the complex dynamic BBB-NO model but also pointing to the necessity to study BBB changes together with NO functions.

Articular cartilage degeneration is a hallmark of degenerative joint diseases, yet its limited regenerative capacity poses significant challenges for tissue engineering. While mesenchymal stem cells (MSCs) are the most commonly employed cell type in cartilage tissue engineering, they exhibit donor variability, restricted expansion capacity, and tendencies toward fibrocartilage formation and hypertrophic differentiation. Human induced pluripotent stem cell-derived chondroprogenitors (iCPs) represent a promising alternative, offering scalable production of developmentally relevant cells with intrinsic chondrogenic commitment. However, their performance within three-dimensional biomaterial scaffolds remains largely unexplored. Here, we compared chondrogenic differentiation of iCPs and MSCs within alginate hydrogels of varying stiffness (0.37 - 4.55 kPa) exhibiting physiologically relevant stress relaxation properties. Intermediate stiffness (2% alginate, ~2.17 kPa) optimally supported chondrogenesis for both cell types. While MSCs differentiated as single cells, iCPs spontaneously self-organized into cartilaginous aggregates without requiring a separate pellet pre-culture step, showing significantly higher hyaline indices and reduced COL10 expression, despite initial low viability in hydrogels. To further enhance chondrogenesis, we functionalized 2% alginate gels with RGD and HAVDI peptides mimicking integrin- and cadherin-mediated signaling. HAVDI/RGD functionalization significantly enhanced hyaline cartilage marker expression in both cell types, with iCPs exhibiting superior matrix composition characterized by elevated aggrecan and SOX9 expression and reduced COL10 and MMP13 compared to MSCs. These findings establish iCPs as a promising cell source for cartilage tissue engineering and disease modeling, particularly within biomaterials integrating mechanical and bioactive cues that recapitulate the native cartilage microenvironment.

Regulatory T cells (Tregs) are key mediators of immune resolution and tissue repair following acute kidney injury (AKI), yet strategies to selectively enrich Tregs within the renal microenvironment remain limited. Interleukin-2 (IL-2) is essential for Treg survival and function, but systemic IL-2 delivery is constrained by a short half-life, narrow therapeutic window, and risk of off-target activation of cytotoxic T cells. Effective Treg-based immunomodulation may therefore require both localized cytokine signaling and targeted recruitment of precursor immune populations. Here, we present a sequential, biomaterial-based immunomodulatory strategy that integrates immune cell recruitment with sustained cytokine delivery. An injectable granular hydrogel physically encapsulating the chemokine CCL21 was used to initially recruit CD4+ T cells to the injured kidney. Subsequently, supramolecular guest-host interactions were leveraged to deliver IL-2 modified with the hydrophobic "guest" moiety adamantane, enabling prolonged local cytokine release over 28 days. In healthy mice, we observed early CD4+ T cell recruitment, followed by a pronounced increase in Treg frequency, consistent with immune reprogramming driven by our sequential delivery system. In a murine model of bilateral ischemia-reperfusion (BiAKI), this injectable biomaterial induced a local increase in the proportion of FOXP3-expressing Tregs and a concomitant decrease in the levels of AKI markers. This two-step immunomodulatory approach minimizes acute immune activation while promoting long-term enrichment of Tregs within the renal microenvironment. Together, these findings demonstrate that granular hydrogels with supramolecular host capability can orchestrate immune recruitment and differentiation in a spatiotemporally controlled manner, providing a versatile immunomodulatory platform for applicability in kidney disease.

Recent efforts to reconstruct critical-sized craniomaxillofacial defects using biomaterials have increasingly sought strategies to accelerate proangiogenic processes immediately after surgical implantation to improve host-graft integration. A class of mineralized collagen scaffolds has recently been identified to promote osteogenesis of bone marrow and adipose-derived mesenchymal stem cells both in vivo and in vitro, motivating efforts to identify strategies to promote osteogenic-angiogenic coupling in order to support regenerative healing. Here, we report the first-time use of a glycoengineering approach to generate a population of engineered ASCs (eASCs) from human adipose-derived stem cells (hASCs). This effort inserts an azido sugar onto the cell membrane that can be selectively targeted using DBCO-tagged biomolecules to selectively boost eASCs activity. We report the effects of azido sugar labeling conditions (concentration and incubation time) on eASCs labeling efficiency. We show proliferation and pro-regenerative gene expression activity of eASCs in mineralized collagen scaffolds are largely similar to conventional hASCs. And we demonstrate that molecular cargo functionalized with dibenzocyclooctyne (DBCO) can be conjugated to azido groups on eASCs via click chemistry. Together, this work shows metabolically labeled eASCs have the potential to be used for selective delivery of activity-inducing bioactive cues to enhance regenerative potential.

Animal-derived surface coatings present the gold standard in stem cell culture. However, they come with major drawbacks such as lot-to-lot inconsistency and an ethically problematic production. Approaches to develop synthetic coating alternatives have largely failed to recapitulate biological functions essential to PSC-self renewal and differentiation. In this study, we have investigated the suitability of a fully synthetic, chemically defined extracellular matrix-mimicking coating named BIOINSTRUCT for hiPSC culture. BIOINSTRUCT was employed during maintenance culture of IMR90-4 cells, as well as directed differentiation of HIHCNi008-A, ACTN2-GFP, and UKKI-011-A cells. BIOINSTRUCT preserved hiPSC characteristics over the experimental period of 10 passages similar to Matrigel. Spontaneous differentiation resulted in the formation of ectodermal, mesodermal and endodermal derivatives, confirming the retention of pluripotency. Directed differentiation on BIOINSTRUCT enabled the generation of neuronal, cardiac and pancreatic endocrine cells. Altogether, our data underscore BIOINSTRUCT as a promising alternative to animal-derived extracellular matrix coatings.

Modeling neural-immune interactions in neurodegenerative and immune-mediated central nervous system (CNS) diseases requires human 3D models that capture cellular diversity and long-term tissue maturation. Here, we present an enhanced human induced pluripotent stem cell (hiPSC)-derived cerebral organoid (CO) platform optimized to mitigate core hypoxia for over 200 days. Timed pro-myelinating cues established organized neuronal layering and progressive axonal myelination through day 140, while vascular fusion yielded assembloids incorporating endothelial structures and microglia. Extended culture (>500-750 days) spontaneously reproduced hallmark features of human CNS aging, including cellular senescence signatures, neuroaxonal loss, hypomyelination, and the autonomous emergence of a neurotoxic astrocyte transcriptional profile in the complete absence of microglia or immune cells. Co-culture with autologous activated peripheral blood mononuclear cells (PBMC) resulted in transient immune infiltration and a pronounced type II interferon response across CNS lineages. High-plex spatial transcriptomics revealed that immune cell infiltration was associated with oligodendrocyte loss and in aged organoids also with downregulated oligodendrocyte myelin gene transcription. While not fully reproducing adult tissue stoichiometry, this platform enables longitudinal modeling of neural-immune crosstalk in age-related and neuroinflammatory CNS disorders.

Neuronal heterogeneity is a defining feature of complex neural circuits, where local differences in firing patterns, activity levels, and temporal dynamics shape information processing and emergent network activity. In adult neurons, this heterogeneity arises from a variety of known and unknown factors including the extracellular environment. Although neurons have been cultured on three-dimensional substrates, the effect of the microenvironments on their firing activity remains poorly understood. Here, we have synthesized two chitosan hydrogel systems seeded with human iPSCs-derived dopaminergic neurons as tunable platforms to control neuronal microenvironments. Both systems were formulated with the ability to incorporate carbon nanotubes (CNT), thus promoting neural interfacing. Calcium imaging combined with computational single-cell analysis demonstrated that supramolecular organization, hydration state, and general physicochemical properties differentially bias neuronal firing dynamics and synchrony leading to the emergence of distinct activity phenotypes despite identical cellular origin. These activity profiles were clustered through K-means and assigned to specific phenotypes including bursting irregular neurons, regular network contributors, or less active/quiescent neurons. Furthermore, CNTs incorporation enhanced local hydrogel compaction, resulting in unique active neuronal phenotypes, highlighting the potential of CNTs to modulate local cellular microenvironments. These findings establish tunable biomaterials as microenvironment contenders for controlling neuronal network state while giving insights on the interplay of different cues in promoting neuronal heterogeneity and functional phenotype relevant to neurodevelopment, neurodegeneration, and disease modelling.

One crucial step during early osteogenesis is the embedding of osteoblasts within a collagen-rich extracellular matrix (osteoid), where they subsequently differentiate into a functional network of osteocytes. However, reconstructing 3D osteocyte networks in vitro remains a major challenge. We recently developed a synthetic microporous hydrogel to support the in vitro culture of 3D bone cell networks. Although matrix biodegradability facilitates cell-material interactions, the influence of micropores on bone tissue morphogenesis and differentiation remains poorly understood. Here, we investigate the effect of cell-scale micropores on bone cell morphogenesis and osteogenic differentiation in vitro. By exploiting polymerization-induced phase separation (PIPS) between 4-arm polyethylene glycol vinyl sulfones and dextran in the presence of hyaluronan, we generated matrix metalloproteinase-sensitive hydrogels with cell-scale micropores. Increasing the dextran concentration enlarged the average pore size from 4 m to 8 m, accompanied by a slight decrease in mechanical stiffness. Following encapsulation within these hydrogels, primary human osteoblasts remained highly viable. Hydrogels with larger pores supported extensive 3D cell network formation, whereas hydrogels with smaller pores exhibited enhanced osteogenic differentiation following 21 days of osteogenic culture. Together, these findings highlight that bone cells are sensitive to microporous physical cues and even minor changes over pore sizes can make an impact on osteocyte-like morphogenesis and differentiation in vitro.

Osteochondral repair requires restoration of zonally organised articular cartilage and subchondral bone, yet translatable implants rarely reproduce this spatial complexity. Here, we developed an off-the-shelf, cell-free multilayer scaffold comprising a superficial 2% (w/v) articular cartilage extracellular matrix (AC-ECM) phase, an intermediate 5% AC-ECM phase and a basal 6% bone ECM (BN-ECM) phase. The scaffold formed continuous interfaces, displayed regionally distinct pore sizes and resisted permanent deformation during cyclic compression. In vitro, constructs seeded with caprine mesenchymal stromal and articular cartilage progenitor cells supported cell expansion and the accumulation of sulfated glycosaminoglycan- and collagen-rich matrix, with regional differences in collagen I, II and X deposition. Following eight weeks of subcutaneous implantation, cell-seeded scaffolds contained more collagenous matrix than unseeded controls, while vascularisation preferentially localised to the BN-ECM phase. The scaffold was then evaluated against empty defects in a caprine osteochondral model for six months. Scaffold treatment significantly improved macroscopic and histological repair, increased chondral tissue fill (~60% versus ~40%), limited cartilage-like tissue extension into the subchondral region and generated a more native-like superficial collagen organisation. Repair tissue further exhibited greater collagen II immunoreactivity, increased ACAN and COL2A1 expression and reduced COL1A2 expression relative to empty defects, although deeper bone repair was not significantly improved. These findings demonstrate that tissue-specific ECM layering can spatially guide endogenous repair and substantially improve cartilage restoration without exogenous cells or growth factors in a clinically relevant large-animal model, while identifying subchondral bone regeneration as the remaining design challenge for complete osteochondral repair.

Osteoarthritis, characterized by cartilage degradation and synovial inflammation, has spurred interest in mechano-piezoelectric bio-hydrogel therapies that can both relieve symptoms and reverse progression. However, current approaches lack sufficient piezoelectric output and dual cartilage/inflammation targeting. To address this, we demonstrated a mechano-piezoelectric peptide hydrogel composed of nanofibers integrating piezoelectric cues with mesenchymal stromal cells (MSCs) recruitment and PIEZO2 mechanosignaling. Molecularly, the hydrogel's seed peptide incorporated four functions: COL2A1 targeting, MMP-13 responsiveness, MSCs homing, and self-assembly. Overexpressed MMP-13 in the osteoarthritis niche triggers gelation, promoting MSCs recruitment and drug retention. Fluorination modulates hierarchical nanofiber assembly, enhancing mechanical and piezoelectric properties, as confirmed by morphological, biophysical, and computational analysis. The trifluoromethyl-modified, 4-octyl itaconate (4-OI) loaded formulation reverses osteoarthritis via PI3K/AKT activation and Wnt/{beta}-catenin suppression, as shown by improved Osteoarthritis Research Society International (OARSI) scores, bone microarchitecture, and cartilage matrix. This synergy of mechano-piezoelectric cues and 4-OI offers a clinically promising strategy for osteoarthritis.

Oxygen (O2) transport is a major limitation in high-density cell culture, particularly as three-dimensional (3D) spheroids are increasingly used to better mimic in vivo tissue environments. Here, we developed a poly(4-methyl-1-pentene) (PMP)-based microwell platform, leveraging its lower molecular absorption and greater mechanical stability than the commonly used O2-permeable polydimethylsiloxane (PDMS), while retaining high O2 permeability. We measured O2 permeability in 50-m-thick PMP membranes and found approximately 200-fold greater O2 permeability than the conventional 1-mm polystyrene culture-plate material. PMP membrane-based microwell plates with 350-m-diameter wells maintained spatial separation of pre-formed hepatocyte spheroids while supplying O2 from the plate bottom, synergistically enhancing spheroid oxygenation. Compared with non-O2-permeable plates, the platform reduced localized hypoxia and hypoxia-associated cell injury while improving spheroid viability and growth. Collectively, PMP-based O2-permeable microwell plates provide a promising, scalable, high-throughput platform for spheroid culture with multiple applications in tissue engineering, drug screening and beyond.

Decellularized extracellular matrix (dECM) scaffolds are increasingly used in regenerative medicine, yet the extent to which processed placental dECM retains properties capable of influencing cellular responses remains unclear. This study combines functional cell assays with deep learning-enabled quantitative imaging to determine how dehydrated placental ECM regulates cellular behavior across multiple human cell lineages. Human dermal fibroblasts, cardiac fibroblasts, and osteoblasts were cultured on dehydrated placental ECM or standard cell culture surfaces and assessed for cell attachment, viability, extracellular matrix production, and nuclear morphology. Placental dECM supported attachment and survival across all three cell types, while Pro-Collagen I Alpha 1 secretion varied by cell lineage relative to negative controls. To identify structural responses associated with scaffold culture, an automated imaging pipeline combining Cellpose-based nuclear segmentation with nuclear morphometric analysis was used to quantify nuclear area, eccentricity, and circularity. Quantitative profiling of hundreds of nuclei revealed scaffold-dependent remodeling of nuclear morphology that was not apparent by conventional microscopy. Cells cultured on placental dECM exhibited reduced nuclear area and increased nuclear eccentricity, while cardiac fibroblasts and osteoblasts showed alterations in nuclear circularity. These lineage-dependent morphological responses demonstrate that placental dECM provides more than a permissive substrate for cell attachment and is associated with measurable changes in cellular architecture following processing. Together, these findings support the biological relevance of processed placental dECM as a regenerative biomaterial and demonstrate the utility of quantitative single-cell morphometric analysis for detecting cell-material interactions that may not be apparent through qualitative imaging alone, guiding the rational design of regenerative therapies.

Articular cartilage damage often progresses to osteoarthritis (OA), a degenerative joint disease characterized by chronic pain, limited mobility, and reduced quality of life. Tissue engineering approaches using poly (ethylene glycol) (PEG)-based hydrogels offer tunable mechanical properties and bioactive functionalization, yet the influence of surface charge on cartilage regeneration remains underexplored. Moreover, recombinant fibroblast growth factor 18 (FGF18) has successfully improved cartilage tissue thickness in clinical trials, but required high dosages and recurring injections may limit compliance. Here, we developed a granular microgel-based platform with bioorthogonally tethered FGF18 to evaluate the interplay of microgel surface charge and growth factor presentation on chondrogenesis. Azide groups were incorporated onto the microgel surfaces to enable site specific FGF18 conjugation across microgel scaffolds with distinct surface charges. When seeded with mesenchymal stromal cells, microgel scaffolds functionalized with FGF18 outperformed their unmodified counterparts, evidenced by higher GAG content, collagen content, and compressive modulus. In a murine microfracture model, anionic and zwitterionic microgel scaffolds tethered with FGF18 increased cartilage regeneration compared to nonionic microgels. We detected increased collagen II content within defects treated with tethered FGF18 microgel scaffolds. This work demonstrates the role of surface charge and growth factor presentation in directing cell behavior and tissue repair, advancing the design of biomaterials for cartilage regeneration. HIGHLIGHTSO_LICovalent tethering of FGF18 to microgel surface enables localized bioactivity C_LIO_LIFGF18 tethered microgel scaffolds enhance extracellular matrix deposition and chondrogenic differentiation of murine mesenchymal stromal cells in vitro C_LIO_LILocalized FGF18 presentation improves cartilage tissue formation and mechanical properties in vivo, with anionic and zwitterionic microgel scaffolds outperforming nonionic scaffolds C_LIO_LIFGF18 presentation is a more potent stimulus than microgel surface charge for cartilage regeneration C_LI

Astrocyte border formation is a conserved neuroprotective response to neural tissue disruption, yet astrocyte border states at implanted biomaterials remain less well characterized than injury responses. Here, we developed the Astrocyte Border Characterization (ABC) Tool, which leverages a shear-thinning, injectable biomaterial to locally deliver astrocyte-specific RiboTag AAVs and small molecule regulators in the mouse striatum, enabling molecular profiling and phenotypic modulation of astrocyte border (AB) cells. Spatially precise delivery of AAV using the ABC Tool yielded enhanced specificity and robust RiboTag expression in AB cells from 7-70 days post injection. Temporal transcriptomic profiling of AB cells revealed predominantly acute, transient changes in genes governing dedifferentiation, proliferation, metabolic reprogramming, and inflammation regulation. Persistent changes accounted for only 14% of regulated genes but involved critical gain of functions in immune regulation and host defense that mirrored astrocyte border responses at chronic CNS injuries. Local delivery of indiscriminate or astrocyte-selective ablation molecules delayed, rather than prevented, border formation, ultimately yielding thicker astrocytes borders with increased inflammation and fibrosis at the biomaterial-tissue interface. Conversely, local delivery of {beta}-hydroxybutyrate (BHB) from the ABC Tool altered key aspects of the transcriptional reprogramming to attenuate chronic astrocyte reactivity and prevent biomaterial contraction without exacerbating inflammation or fibrosis. Our findings establish the ABC Tool as a bioassay for studying and manipulating astrocyte borders at implanted biomaterials and identify focal metabolic regulation as a strategy to modulate AB cell phenotypes and enhance the CNS biocompatibility of biomaterials.

A major challenge in vascular tissue engineering is the development of scalable fabrication methods capable of producing vascular grafts with suitable mechanical properties and hemocompatibility for both preclinical evaluation and future clinical translation. Using a recently developed method to generate acellular vascular grafts from densified collagen hydrogel, we present a proof-of-concept in vivo evaluation of the scalable collagen graft technology. Thick-walled, genipin-crosslinked grafts, representative of a fabrication approach applicable to clinically relevant vessel sizes, were implanted as interposition grafts in the rat abdominal aorta. These grafts supported rapid neointima formation and re-endothelialisation of the luminal surface but developed late thrombosis, highlighting the challenges of evaluating clinically scalable grafts in small-animal models. To enable assessment of the platform in millimetre-scale vessels, we developed a thin-walled (~100 {micro}m), non-crosslinked graft using a constrained densification approach and hyaluronic acid-mediated modulation of collagen fibril assembly to improve compliance matching and hemocompatibility, with the latter providing a matrix-level approach that may complement surface-bound antithrombotic coatings. These grafts remained patent for up to 17 weeks and exhibited neointima formation, re-endothelialisation, and early cellular infiltration into the graft wall. Together, these findings demonstrate in vivo functionality of densified collagen vascular grafts and establish a foundation for future evaluation in larger animal models.

Engineering a transplantable artificial liver requires reconstituting the hepatic lobule, whose densely cellular parenchyma is organized around a hierarchically branched vascular network. Achieving high cellular density and hierarchical vascularization within a single fabrication step remains a major challenge. Here, we present an ultrasonic standing wave (USW)-based strategy that concurrently organizes vascular and parenchymal compartments during a single extrusion. HepG2-endothelial cell aggregates were fabricated as building blocks and suspended in a liver-derived dECM-alginate hydrogel for rapid, ultraviolet-free crosslinking. They were then co-extruded with single endothelial cells through a glass capillary under USW actuation. Due to their size difference, the single cells were focused at the pressure nodes, forming patterns that transitioned from a single supplying line to four microvascular lines. The aggregates instead accumulated at the pressure antinodes, reconstituting the basic microvascular-parenchymal unit of the lobule. Aggregate size increased with seeding density while maintaining uniformity and over 90% viability. The patterned endothelial cells formed perfusable lumina through which 5 um microparticles flowed freely. Compared with hepatocyte-only cultures, the vascularized constructs showed a 1.7-fold increase in urea production, and 2.7- and 6.2-fold increases in CYP1A2 and CYP3A4 expression, respectively. Following implantation into the mouse liver, the scaffolds showed favorable biocompatibility, sustained proliferation over 21 days, and progressive host integration. The smallest blood-containing lumina matured from 26.1 um to 7.9 um in diameter, accompanied by increasing collagen deposition at the graft-host interface. This approach provides a template-free route to hierarchically vascularized hepatic units and a promising step towards implantable liver tissue.

Synthetic hydrogels with bioactive ligands have been utilized to develop 3D models to gain mechanistic insight into how discrete extracellular matrix (ECM) cues direct cell fate. While the RGD motif is ubiquitously present in healthy and diseased tissues, the EDGIHEL (EDG) sequence is present only in the extra domain A-containing fibronectin (ED-A FN), which is transiently deposited in the provisional matrix in the wound bed. Here, we explore the potential of covalently tethered EDG in conjunction with RGD to promote fibroblast-to-myofibroblast transition (FMT). Normal human lung fibroblasts (NHLFs) were maintained in bioorthogonally constructed, hyaluronan-based hydrogel (BOHAGel) with tethered RGD ligands. When EDG was introduced on day 0 during cell encapsulation, cellular expression of Toll-like receptor 4 (TLR4) was upregulated, and a pro-inflammatory matrix remodeling response was observed, but myofibroblast differentiation was not detected. To mimic the transition from a healthy to an injured state, we leveraged the temporal tunability of BOHAGel by supplementing cell culture media with trans-cyclooctene (TCO)-tagged EDG after cells were primed in the RGD environment for 8 days. As the TCO species diffused through the hydrogel, EDG was instantaneously coupled to the network through immobilized tetrazine functionalities. Delayed introduction of profibrotic EDG motifs increased mRNA levels of the myofibroblast marker (ACTA2), ECM proteins (COL1A1, COL3A1, FN1), and transforming growth factor beta1 (TGFbeta1) downstream targets (VEGFA, CTGF), as well as matrix remodeling enzymes (MMP2, TIMP1). These changes were accompanied by the formation of alpha-SMA stress fibers, confirming complete FMT. Delayed EDG conjugation also enhanced and reinforced alpha1 integrin expression. Importantly, removing the RGD signal from the gel failed to induce myofibroblast differentiation. Collectively, our results suggest that FMT depends on ligand identities and the timing of their emergence in engineered matrices.

Transcatheter embolization requires materials that can be steered through tortuous vessels, solidify rapidly in situ, remain clearly visible under fluoroscopy, and, ideally, carry therapeutic cargo without harming tissue. To meet these requirements, we present a fully water-based, two-component PEI-PEG hydrogel delivered through a soft-robotic, microfluidic catheter that keeps the precursors separate until they meet in a millimetre-scale mixing chamber at the tip. Fast amide cross-linking converts the liquid pair into a self-supporting gel within seconds, eliminating organic solvents and preventing catheter blockage. By adjusting precursor ratio and flow regime, the gel's stiffness and viscosity can be tuned over orders of magnitude, with the same chemistry allowing to occlude both high-flow arteries and fragile micro-vessels. The platform was validated in three escalating models. First, in ex-vivo perfused human placenta, the hydrogel filled targeted branches without reflux or fragmentation, demonstrating controlled delivery in clinically relevant vasculature. Next, in three porcine embolizations, splenic, hepatic and ascending pharyngeal arteries, the material achieved stable, selective occlusion with no migration, vasospasm or recanalization, showing seamless compatibility with standard interventional workflows. Finally, in rats bearing orthotopic liver tumours, drug-loaded hydrogel delivered through the hepatic artery concentrated doxorubicin inside tumours while sparing healthy tissue, confirming its potential for precision chemoembolization. These results position the PEI-PEG hydrogel and microfluidic catheter as a unified, image-guided platform that couples robust mechanical occlusion with site-specific drug delivery, offering a biocompatible alternative to current liquid embolics and expanding the therapeutic reach of minimally invasive embolization procedures.

The growing demand for three-dimensional (3D) in vitro models that accurately replicate human tissue to investigate disease mechanisms, enable tissue replacement, and support drug testing is driving the need for more advanced manufacturing platforms. Mesenchymal stem/stromal cells (MSCs) are promising candidates for tissue engineering and stem cell therapies due to their regenerative potential and ethical advantages. However, the reproducible and scalable production of 3D models that truly mimic the human complexity and function poses a major challenge. To address these challenges, we have developed a scalable and semi-automated 3D differentiation process for generating osteogenic microtissue derived from MSCs encapsulated in core-shell capsules (CSCs). To validate material-dependent, osteo-inductive effects of hyaluronic acid (HA), we systematically characterised our encapsulation process with regard to CSC integrity, spheroid formation and viability, as well as diffusion and shear thinning properties. Osteogenic differentiation was performed in static and dynamic culture, using either conventional 6-well plate format or a rotating wall vessel bioreactor. HA-CSCs were compared with inert carboxymethyl cellulose (CMC)-CSCs. In addition, hypoxic culture condition and the implementation of human platelet lysate (hPL) further supported physiological relevance. HA led to the formation of larger spheroids with high cell viability and increased metabolic activity compared to CMC. Calcium phosphate deposition within the extracellular matrix (ECM) and elevated ALP activity confirmed successful osteogenesis in all conditions. Furthermore, both HA and dynamic culture promote osteogenesis, and when combined, synergistically enhance the osteogenic differentiation. In conclusion, we present a semi-automated, high-throughput and xeno-free platform process for the 3D differentiation of MSCs towards osteogenic lineage. By integrating HA, hypoxia and dynamic culture conditions, this capsule-based system enhances the physiological relevance, enabling advanced in vitro disease modelling, drug testing, and scalable microtissue generation while reducing reliance on animal models.