Advanced Materials

We demonstrate the formulation of advanced functional 3D printing inks that prevent the formation of bacterial biofilms in vivo. Starting from polymer libraries, we show that a biofilm resistant object can be 3D printed with the potential for shape and cell instructive function to be selected independently. When tested in vivo, the candidate materials not only resisted bacterial attachment but drove the recruitment of host defences in order to clear infection. To exemplify our approach, we manufacture a finger prosthetic and demonstrate that it resists biofilm formation – a cell instructive function that can prevent the development of infection during surgical implantation. More widely, cell instructive behaviours can be ‘dialled up’ from available libraries and may include in the future such diverse functions as the modulation of immune response and the direction of stem cell fate.Competing Interest StatementThe authors have declared no competing interest.View Full Text

Biomechanical contributions of the ECM underpin cell growth and proliferation, differentiation, signal transduction, and other fate decisions. As such, biomaterials whose mechanics can be spatiotemporally altered - particularly in a reversible manner - are extremely valuable for studying these mechanobiological phenomena. Herein, we introduce a poly(ethylene glycol) (PEG)-based hydrogel model consisting of two interpenetrating step-growth networks that are independently formed via largely orthogonal bioorthogonal chemistries and sequentially degraded with distinct bacterial transpeptidases, affording reversibly tunable stiffness ranges that span healthy and diseased soft tissues (e.g., 500 Pa - 6 kPa) alongside terminal cell recovery for pooled and/or single-cell analysis in a near "biologically invisible" manner. Spatiotemporal control of gelation within the primary supporting network was achieved via mask-based and two-photon lithography; these stiffened patterned regions could be subsequently returned to the original soft state following sortase-based secondary network degradation. Using this approach, we investigated the effects of 4D-triggered network mechanical changes on human mesenchymal stem cell (hMSC) morphology and Hippo signaling, as well as Caco-2 colorectal cancer cell mechanomemory at the global transcriptome level via RNAseq. We expect this platform to be of broad utility for studying and directing mechanobiological phenomena, patterned cell fate, as well as disease resolution in softer matrices. TOC DescriptionBiomaterials that can dynamically change stiffnesses are essential in further understanding the role of extracellular matrix mechanics. Using independently formulated and subsequently degradable interpenetrating hydrogel networks, we reversibly and spatiotemporally trigger stiffening/softening of cell-laden matrices. Terminal cell recovery for pooled and/or single-cell analysis is permitted in a near "biologically invisible" manner. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/588191v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@d89309org.highwire.dtl.DTLVardef@9d6dc0org.highwire.dtl.DTLVardef@19065e6org.highwire.dtl.DTLVardef@1120aec_HPS_FORMAT_FIGEXP M_FIG C_FIG

Bioadhesive materials and patches are promising alternatives to surgical sutures and staples. However, many existing bioadhesives do not meet the functional requirements of current surgical procedures and interventions. Here we present a translational patch material that exhibits: (1) instant adhesion to wet tissues (2.5-fold stronger than Tisseel, an FDA-approved fibrin glue), (2) ultra-stretchability (stretching to >300% its original length without losing elasticity), (3) compatibility with rapid photo-projection (<2 min fabrication time/patch), and (4) ability to deliver therapeutics. Using our established procedures for the in silico design and optimization of anisotropic-auxetic patches, we create next generation patches for instant attachment to wet and dry tissues while conforming to a broad range of organ mechanics ex vivo and in vivo. Patches coated with exosomes demonstrate robust wound healing capability in vivo without inducing a foreign body response and without the need for patch removal that can cause pain and bleeding. We further demonstrate a new single material-based, void-filling auxetic patch designed for the treatment of lung puncture wounds. TeaserWe demonstrate a sticky and highly elastic patch with conforming designs for dynamic organ repair.

In vivo genetic engineering of T cells could overcome the logistical, biological, and safety challenges of ex vivo modification, but effective and safe delivery systems remain limited by a lack of cellular specificity. Here, we developed aptamer-functionalized lipid nanoparticles (LNPs) for targeted mRNA delivery to CD4+ T cells, employing both a validated CD4-binding aptamer (Apt62) and novel aptamers generated using our proprietary transformer-based AI language model, AptaBLE. LNPs formulated with ionizable lipid SM102 or MC3 and conjugated with aptamers at controlled densities were physiochemically characterized and assessed for binding, in vitro transfection, in vivo biodistribution, and safety evaluation. Aptamer-functionalized LNPs demonstrated selective nanomolar binding to recombinant CD4, achieved enhanced transfection of CD4+ versus CD4- T cells in vitro, and significantly enriched mRNA delivery to immune-rich tissues in vivo, achieving up to 70-fold spleen signal enhancement with SM102 formulations compared to non-targeted controls, while maintaining suitable safety profiles. Overall, these findings demonstrate aptamer-functionalized LNPs, augmented by AI-guided aptamer design, as a tunable, non-immunogenic platform for in vivo T cell engineering. HighlightsO_LIAptamer-functionalized LNPs enable selective mRNA delivery to CD4+ immune cells rich organs. C_LIO_LIAptamer-functionalized LNPs maintain a favorable systemic safety profile in vivo. C_LIO_LIAI-guided AptaBLE platform generated functional aptamers validated in nanoparticle delivery. C_LI

Microbes are increasingly utilized as living therapeutic vehicles, yet their uncontrolled dissemination in the body has long remained a roadblock to clinical development. Physical containment, while widely used for mammalian cells, remains largely unattainable due to eventual bacteria escape. Here, we present an implantable material platform that encapsulates and confines bacteria, wherein synthetically engineered microbes produce therapeutic payloads from within. To prevent microbial escape, we developed a hydrogel scaffold with dual mechanical features: high stiffness to regulate bacterial proliferation and high toughness to resist material fracture under physiological stress. This design achieved complete bacterial containment for over six months and withstood multiple forms of mechanical loading that otherwise caused catastrophic material failure. By genetically engineering embedded bacteria, we endowed the material with environmental sensing and on-demand therapeutic release capabilities and demonstrated autonomous treatment in a murine prosthetic joint infection model. This multimodal strategy provides a safe and generalizable framework for deploying microbial medicines in vivo and supports their use as autonomous drug depots across a range of disease settings.

Material- and cell-based technologies such as engineered tissues hold great promise as human therapies. Yet, the development of many of these technologies becomes stalled at the stage of pre-clinical animal studies due to the tedious and low-throughput nature of in vivo implantation experiments. We introduce a plug and play in vivo screening array platform called Highly Parallel Tissue Grafting (HPTG). HPTG enables parallelized in vivo screening of 43 three-dimensional microtissues within a single 3D printed device. Using HPTG, we screen microtissue formations with varying cellular and material components and identify formulations that support vascular self-assembly, integration and tissue function. Our studies highlight the importance of combinatorial studies that vary cellular and material formulation variables concomitantly, by revealing that inclusion of stromal cells can "rescue" vascular self-assembly in manner that is material-dependent. HPTG provides a route for accelerating pre-clinical progress for diverse medical applications including tissue therapy, cancer biomedicine, and regenerative medicine.

Engineering physiologically relevant 3D microenvironments is critical for studying cell behavior and advancing regenerative medicine. We present a new hybrid scaffold, fabricated via MultiPhoton Lithography (MPL), that integrates synthetic polymers (BisSR/CEA) with methacrylated collagen type I (Coll-MA) for single-cell enclosure and long-term culture. This is the first demonstration of a 3D MPL-printed biodegradable scaffold that mimics bone-like stiffness and allows spatially controlled, biodegradation-driven remodeling. The nanoscale feature size and mechanical properties are validated using Atomic Force Microscopy (AFM), while the nanoscale bioactivity of the scaffold is confirmed through Single-Molecule Localization Microscopy (SMLM). We track vinculin, a focal adhesion protein, with single-molecule resolution during mesenchymal stem cell (MSC) expansion and osteogenic differentiation. A new finding is time-dependent axial migration of vinculin clusters, independent of scaffold composition. Despite similar mechanosensing profiles, hybrid scaffolds significantly enhance osteogenic marker expression (collagen I, osteocalcin), revealing that scaffold bioactivity and geometry, not stiffness alone, direct stem cell fate. Cell expansion is highly dependent on scaffold composition, showing a biodegradation-driven remodeling over time. This platform offers a new tool to study cell-matrix interactions at the single-cell and single-molecule level and holds promise for Organ-on-Chip systems (e.g. bone-cartilage interface models), and personalized regenerative therapies.

The endothelialization of organ-on-chip platforms and vascular implants is often limited by slow cell attachment and unstable monolayer formation. This work presents a scalable workflow that imprints micro- and nano-gratings into elastin-like recombinamer (ELR)-based hydrogels, enabling rapid endothelial cell capture and accelerating monolayer formation within 14 days. Three gelatin-ELR formulations are engineered, with {superscript 1}H-NMR confirming incorporation of sequences designed to modulate bioactivity (ELR1: inert; ELR2: uPA-responsive; ELR3: RGD-adhesive). ELR incorporation generates fibrillar microstructures and enhances mechanical performance, yielding elastic-dominant networks suitable for high-fidelity pattern transfer and stable culture. Using this library, the combined effects of ELR bioactivity and groove geometry on human iPSC-derived endothelial cells (iPSC-ECs) are systematically evaluated. In a 15-minute attachment assay, patterned ELR composites markedly improve cell retention compared to gelatin, with ELR2 on [~]350 nm and [~]4 {micro}m grooves performing best, consistent with controlled, cell-mediated interfacial remodeling. This early advantage persists, as ELR2 and ELR3 hydrogels support rapid alignment and reach confluence by day 14, whereas gelatin remains sub-confluent. Cytoskeletal analysis confirms F-actin alignment. By combining enhanced early capture with protease-regulated remodeling, ELR2 identifies a favorable design window. These results establish a materials design framework linking programmable ELR chemistry with surface topography to engineer endothelial interfaces, providing a versatile platform for vascular biomaterials and microphysiological systems.

To treat acute myocardial infarction immediately after reperfusion, we previously engineered an intravascularly infusible decellularized extracellular matrix (iECM) biomaterial that exerts immunomodulatory and pro-reparative effects. However, the impact of the heterogeneous contents of iECM on infarct localization and downstream biological function is unknown. Using liquid chromatography, iECM is separated into a high molecular weight (MW) and low MW component. Mass spectrometry confirms compositional similarity, while biochemical assays and transmission electron microscopy highlight differences in biochemical features and structure, revealing a nanofibrillar high MW component and a globular peptide low MW. Quartz crystal microbalance studies show binding of each component to basal lamina ECM proteins and endothelial cell surface receptors under flow, demonstrating the specificity of ECM biomaterials to permeable vasculature. In vivo, the low MW component reduces vascular permeability, while neither component alone achieves the retention levels of complete iECM. Using single-nucleus RNA sequencing to probe bioactivity, both components elicited comparable angiogenic, immunomodulatory, and pro-reparative transcriptional programs. These findings illustrate that highly coupled materials and biological characterization uncover fundamental behaviors and properties of iECM biomaterials. Additionally, we show the unique binding behavior of iECM to the gaps of permeable vasculature, which could be exploited for future nanomaterial design.

We present a proof-of-concept platform in which amyloids are displayed on the surface of engineered Bacillus subtilis spores for bioengineered materials. Amyloids possess high tensile strength, elasticity, and tunable assembly, but their use is limited by inaccessible native sources and low-yield or toxic heterologous expression. Here, spores were engineered to display the native amyloid TasA and Humboldt squid suckerins 9 and 10 as fusions to the spore coat protein CotY. Amyloid production and fibril formation were confirmed by Western blot and X-34 staining, and quantitative analysis indicated mg/L-level yields. Atomic force microscopy revealed altered stiffness and surface ultrastructure, and incorporation of amyloid-displaying spores into resin-based 3D printing modified tensile strength. These findings highlight spore-based amyloid display as a scalable, modular platform for materials applications, leveraging established industrial spore production.

Biomaterial scaffolds have served as the foundation of tissue engineering and regenerative medicine. However, scaffold systems are often difficult to scale in size or shape in order to fit defect-specific dimensions, and thus provide only limited spatiotemporal control of therapeutic delivery and host tissue responses. Here, a lithography-based three-dimensional (3D) printing strategy is used to fabricate a novel miniaturized modular LEGO-like cage scaffold system, which can be assembled and scaled manually with ease. Scalability is based on an intuitive concept of stacking modules, like conventional LEGO blocks, allowing for literally thousands of potential geometric configurations, and without the need for specialized equipment. Moreover, the modular hollow-cage design allows each unit to be loaded with biologic cargo of different compositions, thus enabling controllable and easy patterning of therapeutics within the material in 3D. In summary, the concept of miniaturized cage designs with such straight-forward assembly and scalability, as well as controllable loading properties, is a flexible platform that can be extended to a wide range of materials for improved biological performance. TOC3D printed LEGO-like hollow microcages can be easily assembled, adjoined, and stacked-up to suit the complexity of defect tissues; aid spatial loading of cells and biomolecules; instruct cells migration three-dimensionally; and facilitate cell invasion and neovascularization in-vivo, thus accelerating the process of tissue healing and new tissue formation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/974204v1_ufig1.gif" ALT="Figure 1"> View larger version (88K): org.highwire.dtl.DTLVardef@1a97a72org.highwire.dtl.DTLVardef@1a60fbeorg.highwire.dtl.DTLVardef@1538f13org.highwire.dtl.DTLVardef@d6345b_HPS_FORMAT_FIGEXP M_FIG C_FIG

Metastatic breast cancer (BC) is the main cause of cancer-related death in women. Accumulating evidence highlights the prominent changes within the lung metastatic niche, resulting in stiffening of the extracellular matrix (ECM). The prevailing concept of cancer evolution encompasses the acquisition of beneficial traits as a consequence of genomic instability, yet it remains elusive to what extend altered lung ECM mechanics feed into this. To investigate this, a tunable 3D bioengineered model, capturing the biophysical and biochemical characteristics of the BC metastatic lung niche is developed. Porcine derived lung decellularized ECM (dECM), combined with norbornene and tetrazine modified click-crosslinkable alginate, recapitulates composition and mechanics of healthy soft (3 kPa) and metastatic stiff (13 kPa) lung niches. Label-free optical microscopy further validates the microarchitectural resemblance between resulting matrices and human lung metastasis samples. Encapsulation of MDA-MB-231 and MCF7 cells reveals that stiffer matrices promote BC cluster growth and DNA damage, indicated by yH2AX, independent of BC subtype. Moreover, this platform is compatible with patient derived cells, which remain viable for 14 days. These findings underscore the critical role of tissue mechanics in regulating BC metastasis progression and demonstrate the utility of the herein developed tunable, physiologically relevant platform for patient-based models. Table of Content O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=180 SRC="FIGDIR/small/698349v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@19a164dorg.highwire.dtl.DTLVardef@12e1c1aorg.highwire.dtl.DTLVardef@371378org.highwire.dtl.DTLVardef@1df3ddb_HPS_FORMAT_FIGEXP M_FIG C_FIG Fabrication of 3D bioengineered breast cancer (BC) lung metastasis niche to investigate how tissue mechanics modulate cancer evolution. Tunable hybrid biomaterials recapitulate the mechanical and biochemical characteristics of healthy soft and diseased stiff lung. Within these niches, stiffness promotes enhanced BC cluster growth and genomic instability. Biocompatibility with patient-derived BC cells, opens opportunities for drug testing platforms for personalized medicine.

Rapid in situ bioprinting on complex, human-scale anatomical surfaces remain a key challenge for clinical translation. Here, we present a gravity-independent, conformal bioprinting strategy using bi-phasic granular bioink and multinozzle printheads capable of adapting to arbitrary surface curvatures. The bioink comprised of jammed gelatin microgels suspended in a fibrinogen matrix exhibits yield-stress behavior to maintain shape fidelity after extrusion while supporting cell viability and proliferation. Two monolithic multinozzle printhead architectures with identical bioink delivery networks were evaluated: (1) a rigid configuration for handheld bioprinting and (2) a soft robotic variant capable of real-time curvature adaptation via pneumatic actuation. Microgravity experiments aboard a parabolic flight confirmed successful bioink deposition under [~]0 g conditions. A ladder-rung microfluidic architecture ensured uniform bioink delivery across printhead nozzles, improving deposition consistency. In situ bioprinting on anatomical facial phantoms confirmed conformal, high-throughput (deposition at 20 mm{middle dot}s-1) deposition of bioink over physiologically relevant curvatures, both with and without cells. Cell-laden constructs retained >85% cell viability post-printing and supported proliferation. This work introduces a scalable bioprinting platform suitable for clinical, remote, and deep-space environments, enabling autonomous tissue fabrication. The curvature-adaptive printhead advances current in situ bioprinting capabilities, facilitating the generation of personalized grafts with complex anatomical geometries.

The human vasculature is a complex, multiscale system comprising hierarchical networks of macroscale to microscopic vessels. Existing in vitro fabrication techniques often fail to bridge these disparate scales, as high-resolution methods like multiphoton ablation are too slow for replicating larger vessels, while 3D printing lacks the resolution for fine microscale features. Here, we report a "twisted wire templating" strategy capable of generating perfusable bifurcating hydrogel networks that seamlessly transition from the macro- to the micro-scale (2.3 mm to 140 {micro}m) through seven orders of bifurcations. By optimizing wire-twisting geometries and polyurethane dip-coating, we overcame instability-driven bead formation to ensure replication fidelity across the networks. Fabrication rigs were reconfigured from existing 2D planar layouts to 3D reconfigurable architectures to better replicate 3D vessel geometries which simultaneously reducing the laboratory footprint and fabrication times by 47%. Using a Taguchi orthogonal array, we further optimized surface chemistry and hydrogel composition to inhibit structural failure during template extraction, resulting in fully patent, perfusable networks. This method provides a robust, low-cost, and scalable foundation for creating physiologically representative vascular models for investigating multiscale disease mechanisms and organ-level tissue engineering.

Anisotropic biological tissues contain hierarchical complexity from the nano to macro length scales. While novel fabrication strategies have advanced the creation of biomimetic architectures, most rely on biologically derived polymers that possess inherent batch-to-batch variability. Here, we fabricate omnidirectional anisotropic nanofibrous hydrogels using synthetic, self-assembling MultiDomain Peptides (MDPs). Using support bath-assisted extrusion 3D printing, MDP hydrogels are created with control over nanometer-scale fibrous alignment, ~150 {micro}m-scale print resolution, and centimeter-scale 3D architecture. Further, scaffold anisotropy is tuned by adjusting the ionic strength of the support bath, allowing fiber alignment to be decoupled from extrusion shear force and the ink used. Applying these hydrogels to in vitro tissue engineering, fabricated anisotropic hydrogels are shown to guide the alignment of multiple cell types within complex 3D prints. Furthermore, the gels are demonstrated to support the growth of human embryonic stem cell-derived cardiomyocytes into functional tissue. Collectively, this work introduces a platform for engineering anisotropic peptide hydrogels with hierarchical complexity, offering broad potential for bottom-up fabrication of functional human tissues in vitro.

Hydrogel biomaterials offer great promise for 3D cell culture and therapeutic delivery. Despite many successes, challenges persist in that gels formed from natural proteins are only marginally tunable while those derived from synthetic polymers lack intrinsic bioinstructivity. Towards the creation of biomaterials with both excellent biocompatibility and customizability, recombinant protein-based hydrogels have emerged as molecularly defined and user-programmable platforms that mimic the proteinaceous nature of the extracellular matrix. Here, we introduce PhoCoil, a dynamically tunable recombinant hydrogel formed from a single protein component with unique multi-stimuli responsiveness. Physical crosslinking through coiled-coil interactions promotes rapid shear-thinning and self-healing behavior, rendering the gel injectable, while an included photodegradable motif affords on-demand network dissolution via visible light. PhoCoil gel photodegradation can be spatiotemporally and lithographically controlled in a dose-dependent manner, through complex tissue, and without harm to encapsulated cells. We anticipate that PhoCoil will enable new applications in tissue engineering and regenerative medicine.

Biomaterials with highly tunable mechanical properties and tissue-mimetic structural features are critical for diverse biomedical applications. Photopolymerizable citrate-based polymers (CBP), such as methacrylate polydiolcitrate (mPDC), enable high-resolution fabrication of biodegradable scaffolds via light-based 3D printing for regenerative engineering. However, mPDC scaffolds typically exhibits substantial brittleness due to the formation of highly crosslinked and heterogeneous polymer network, an intrinsic limitation of many acrylate-based polymers, thereby restricting their use across a broad range of tissue types. Herein, we report facile network-engineering strategies to modulate crosslinking density and network topology of CBPs through the incorporation of acrylate-based reactive diluents and/or a thiol-based chain transfer agent, 3,6-dioxa-1,8-octanedithiol (DOD). These approaches enabled significantly improved and broadly tunable mechanical properties, with Youngs modulus spanning 6.8-134 MPa, ultimate tensile strength ranging from 1.8 MPa to 18 MPa, and strain at break varying from 14% to 61%. Notably, incorporation of isobornyl acrylate (IBOA) alone significantly enhanced toughness, yielding a 3.6-fold increase in Youngs modulus (50 MPa vs. 14 MPa) and a 2.8-fold increase in strain at break (39% vs. 14%). Moreover, combined incorporation of IBOA and DOD remarkably improved ductility, achieving a 4-fold increase in strain at break to 61% while maintaining comparable stiffness. All mPDC composites exhibited tunable biodegradability, good cytocompatibility, and excellent 3D printability. Using these composite inks, 3D-printed meniscus scaffolds supported the human chondrocyte growth and fibrochondrogenic matrix deposition, while 3D-printed vascular stents supported endothelial monolayer formation. Collectively, this study establishes a versatile photopolymerizable citrate-based biomaterial platform with broadly tunable mechanical performance, controllable biodegradability, good cytocompatibility, and high printability, offering strong potential for customized biomedical applications ranging from load-bearing to soft tissue engineering.

The tissue microenvironment is comprised of a complex assortment of multiple cell types, matrices, membranes and vessel structures. Emulating this complex and often hierarchical organization in vitro has proved a considerable challenge, typically involving segregation of different cell types using layer-by-layer printing or lithographically patterned microfluidic devices. Bioprinting in granular materials is a new methodology with tremendous potential for tissue fabrication. Here, we demonstrate the first example of a complex tumor microenvironment that combines direct writing of tumor aggregates, freeform vasculature channels, and a tunable macroporous matrix as a model to studying metastatic signaling. Our photocrosslinkable microgel suspensions yield local stiffness gradients between particles and the intervening space, while enabling the integration of virtually any cell type. Using computational fluid dynamics, we show that removal of a sacrificial Pluronic ink defines vessel-mimetic channel architectures for endothelial cell linings. Pairing this vasculature with 3D printing of melanoma aggregates, we find that tumor cells within proximity migrated into the prototype vasculature. Together, the integration of perfusable channels with multiple spatially defined cell types provides new avenues for modelling development and disease, with scope for fundamental research and drug development.

In tissues where the vasculature is either lacking or abnormal, biomaterials can be designed to promote vessel formation and enhance tissue repair. In this work, we independently tune the microstructure and bioactivity of microporous annealed particle (MAP) scaffolds to guide cell patterning in 3D and promote de novo assembly of endothelial progenitor-like cells into vessels. We implement both in silico characterization and in vitro experimentation to elucidate an optimal scaffold formulation for vessel formation. We determine that MAP scaffolds with pore volumes on the same order of magnitude as cells facilitate cell growth and vacuole formation. We achieve spatial control over cell spreading by incorporating adhesive microgels in well-mixed, heterogeneous MAP scaffolds. While we demonstrate that integrin engagement is the primary driver of network formation in these materials, introducing adhesive microgels loaded with heparin nanoparticles leads to the formation of vascular tubes after 3 days in culture. We then show in vivo that this unique scaffold formulation enhances vessel maturation in a wound healing model and instructs differential vascular patterning in the tumor microenvironment. Taken together, this work determines the optimal microstructure and ligand presentation within MAP scaffolds that lead to vascular constructs in vitro and facilitate neovascularization in vivo.

Recreating 3D bone formation in vitro without biochemical inducers remains a longstanding challenge in preclinical testing. We present a scalable, bioinstructive platform based on polylactic acid microparticles with controlled dimpled surface features that direct mesenchymal stem cell differentiation through endogenous topography-mediated mechanotransduction, establishing a mechanistically validated, additive-free platform. These 3D topographical cues drive cytoskeletal reorganisation and induce osteogenesis via canonical Hedgehog signalling. RNA-Seq revealed early significant upregulation of cytoskeletal components and osteochondral transcription factors, including runt-related transcription factor 2 (RUNX2) and SRY-box transcription factor 9 (SOX9), followed by activation of the insulin growth factor-II pathway and osteogenic commitment. To demonstrate translational potential, two-photon polymerisation lithography was employed to engineer precisely-patterned 3D topographies, inducing graded GLI1 expression without added soluble cues. This establishes a modular, versatile platform for stem cell engineering, offering a topography-driven, non-genetic analogue to mechanogenetics with broad utility for regenerative medicine and human-relevant development of bone models. O_FIG O_LINKSMALLFIG WIDTH=162 HEIGHT=200 SRC="FIGDIR/small/664383v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@1b06d7dorg.highwire.dtl.DTLVardef@1e464e1org.highwire.dtl.DTLVardef@19b1299org.highwire.dtl.DTLVardef@1e21f89_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG