Advanced Healthcare Materials

Critical-sized bone defects and implant-associated complications are often exacerbated by chronic inflammation, which compromises tissue repair and implant integration. Mesenchymal stromal cell (MSC)-derived extracellular vesicles have emerged as promising immunomodulatory nanotherapeutics; however, their clinical translation remains constrained by low yield, heterogeneity, and poor scalability. Here we present a bioengineered MSC-derived nanoghosts platform designed to overcome these translational barriers while enabling tunable osteoimmunomodulatory function. By coupling high-yield nanoghost fabrication with biomimetic MSC conditioning, we demonstrate that oxygen tension (5 or 21% O2) and 3D culture substrates (5 or 15 wt-% GelMA) can reprogram MSC immunophenotype. Nanoghosts generated under hypoxic and 3D conditions displayed enriched anti-inflammatory cargo, preserved MSC viability under inflammatory stress, and partially rescued osteogenic mineralization in the presence of pro-inflammatory cytokines. Together, these findings showcase MSC nanoghosts as scalable and bioactive immunoregulatory nanotherapeutic capable of modulating immune-bone crosstalk, providing a translational strategy to mitigate inflammation-driven impairment of bone regeneration and implant integration. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/724218v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1551655org.highwire.dtl.DTLVardef@12d3371org.highwire.dtl.DTLVardef@8c50bborg.highwire.dtl.DTLVardef@834a8_HPS_FORMAT_FIGEXP M_FIG C_FIG

Pediatric cancer survivors treated with gonadotoxic chemotherapy or radiation face lifelong premature ovarian insufficiency (POI), leading to elevated risk of cardiovascular disease, osteoporosis, and metabolic dysfunction. Pharmacological hormone replacement therapy (HRT) cannot replicate the pulsatile, bidirectional signaling of the hypothalamic-pituitary-gonadal (HPG) axis, leaving a critical therapeutic gap. Immune-isolating hydrogel capsules offer a promising strategy for the implantation of donor ovarian tissue without immunosuppression yet they require optimization for human applications. Here, we engineer a microporous immune-isolating capsule by incorporating thermosensitive gelatin microgels as sacrificial porogens. Microfluidic fabrication yielded monodisperse microgels that dissolved at 37{degrees}C generating disconnected micropores within a non-degradable poly(ethylene glycol) (PEG) matrix. Critically, the diffusion of FSH-scale analogs (40 kDa) increased by almost two-fold through the microporous capsules relative to nanoporous controls, while antibody-scale molecules (150 kDa) were blocked, demonstrating size-discriminating permeability. In ovariectomized mice implanted with encapsulated ovarian xenografts for 20 weeks, microporous capsules restored dynamic HPG-axis signaling evidenced by elevated levels of estradiol and progesterone, FSH suppression, and fluctuating hormone levels that resembled physiological patterns. Microporosity also improved graft viability, increasing stromal cellularity and reducing follicular apoptosis. These findings support microporous immune-isolating capsules as a platform for physiologically authentic therapy for POI.

Human mesenchymal stem cells (hMSCs) are widely represented in ongoing regenerative medicine clinical trials due to their ease of autologous implantation. In bone regeneration, crosstalk between macrophages and hMSCs is critical with macrophages playing a key role in the recruitment and differentiation of hMSCs. However, engineered biomaterials able to both direct hMSC fate and modulate macrophage phenotype have not yet been identified. A novel combinatorial chemistry-microtopography screening platform, the ChemoTopoChip, is used to identify materials suitable for bone regeneration by screening with human immortalized mesenchymal stem cells (hiMSCs) and human macrophages. The osteoinduction achieved in hiMSCs cultured on the "hit" materials in basal media is comparable to that seen when cells are cultured in osteogenic media, illustrating that these materials offer a materials-induced alternative in bone-regenerative applications. These also exhibit immunomodulatory effects, concurrently polarizing macrophages towards a pro-healing phenotype. Control of cell response is achieved when both chemistry and topography are recruited to instruct the required cell phenotype, combining synergistically. The large library of materials reveals that the relative roles of microtopography and material chemistry are similar, and machine learning identifies key material and topographical features for cell-instruction.

Glycosylation regulates immune and neural functions within the central nervous system (CNS), yet biomaterials rarely leverage glycans due to their structural complexity. Polysialic acid (PSA), comprising 2,8-linked sialic acid residues, is a promising candidate owing to its potent immunomodulatory interactions with inhibitory Siglec receptors. Systematic screening of multiple sialic acid derivatives identifies PSA as uniquely effective in inducing anti-inflammatory polarization of bone marrow-derived macrophages (BMDMs). Based on these findings, an injectable microporous annealed particle (MAP) scaffold presenting PSA covalently via its reducing end (MAP-PSA) is engineered, recapitulating physiological glycan orientation. MAP-PSA exhibits robust mechanical properties, stable glycan immobilization, and resistance to enzymatic degradation. Using ischemic stroke as a CNS injury model, MAP-PSA significantly reduces neutrophil infiltration and inflammatory activation while enhancing reparative macrophage and microglial phenotypes. These immunomodulatory effects persist into subacute stages, characterized by sustained reductions in inflammation and enhanced microglial homeostasis. Overall, MAP-PSA scaffolds demonstrate a novel therapeutic paradigm for CNS injuries such as stroke, with translational potential for broader neuroinflammatory and regenerative applications.

Demyelinating diseases are a group of complex neurodegenerative disorders characterized by damage to the myelin, the protective sheath that insulates and supports efficient nerve signal conduction. Such a loss of myelin causes the formation of lesions not only in the brain but also often in the spinal cord (SC). Despite the high prevalence of SC lesions among patients, existing models mostly focus on those in the brain, inadequately capture the unique anatomical and physiological features of SC pathology. In this study, we developed a robust, reproducible in vitro model of SC demyelination by combining microwell technology and piezoelectric scaffolds to engineer human neural stem cell (hNSC)-derived nerve tissues featuring aligned, myelinated, extended axons up to 2000 {micro}m in length. We utilized distinct chemical treatments to induce demyelination with or without axonal degeneration: a cuprizone cocktail, a copper chelator combined with inflammatory cytokines, and lysophosphatidylcholine (LPC). Electrophysiological assessments validated the physiological relevance of our model, demonstrating impaired signal transmission and neural connectivity akin to in vivo demyelination pathology. Our versatile platform thus provides a valuable tool for elucidating SC demyelination pathophysiology and exploring potential therapeutic interventions.

Glioblastoma (GBM) tumor cells are found in the perivascular niche microenvironment and are believed to associate closely with the brain microvasculature. However, it is largely unknown how the resident cells of the perivascular niche, such as endothelial cells, pericytes, and astrocytes, influence GBM tumor cell behavior and disease progression. We describe a three-dimensional in vitro model of the brain perivascular niche developed by encapsulating brain-derived endothelial cells, pericytes, and astrocytes in a gelatin hydrogel. We show that pericytes and astrocytes explicitly contribute to vascular architecture and maturation. We use co-cultures of patient-derived GBM tumor cells with brain microvascular cells to identify a role for pericytes and astrocytes in establishing a perivascular niche environment that modulates GBM cell invasion, proliferation, and therapeutic response. Engineered models provides unique insight regarding the spatial patterning of GBM cell phenotypes in response to a multicellular model of the perivascular niche. Critically, we show that engineered perivascular models provide an important resource to evaluate mechanisms by which inter- cellular interactions modulate GBM tumor cell behavior, drug response, and provide a framework to consider patient-specific disease phenotypes.

Building brain foundation models to capture the underpinning neural dynamics of human behavior requires large functional neural datasets for training, which current implantable Brain-Computer Interfaces (iBCIs) cannot obtain due to the instability of rigid materials in the brain. How can we achieve high-density neural recordings with wide brain region access at single-neuron resolution, while maintaining long-term stability? In this study, we present a novel approach to overcome these trade-offs by introducing Fleuron, a family of ultrasoft, ultra-low-k dielectric materials compatible with thin-film scalable microfabrication techniques. We successfully integrate up to 1,024 channels within a single minimally invasive Fleuron depth electrode. The combination of the novel implant material and geometry enables single-unit level recordings for 18 months in rodent models, and achieves a large number of units detected per electrode across animals. 128-channel Fleuron probes, that cover 8x larger tissue volume than state-of-the-art polyimide counterparts, can track over 100 single-units over months. Stability in neural recordings correlates with reduced glial encapsulation compared to polyimide controls up to 9-month post-implantation. Fleuron probes are integrated with a low-power, mixed-signal ASIC to achieve over 1,000 channels electronic interfaces and can be safely implanted in depth using minimally invasive surgical techniques via a burr hole approach without requiring specialized robotics. Fleuron probes further create a unique contrast in clinical 3T MRI, allowing for post-operative position confirmation. Large-animal and ex vivo human tissue studies confirm safety and functionality in larger brains. Finally, Fleuron probes are used for the first time ever intraoperatively during planned resection surgeries, confirming in-human usability, and demonstrating the potential of the technology for clinical translation in iBCIs.

Injury to the central nervous system (CNS) causes inflammation, cell death, and glial scar formation that inhibits tissue repair. Injectable hydrogels modified with extracellular matrix (ECM)-derived peptides can provide biochemical cues to promote neural tissue repair and serve as a vehicle to deliver therapeutics across the blood-spinal/blood-brain barrier in a minimally invasive manner. We developed an injectable hydrazone crosslinked hyaluronic acid-alginate (HA-Alg) hydrogel for neural tissue repair. We fabricated hydrogels with a range of polymer concentrations and evaluated their physicochemical properties to identify formulations that mimic the stiffness and viscoelastic properties of the CNS tissue environment. Hyaluronic acid was further modified with ECM-derived, cell-adhesive peptides (RGD and IKVAV) to enhance neuronal adhesion and viability. To evaluate the therapeutic potential of our hydrogel platform, we embedded mouse embryonic stem cell aggregates and differentiated them toward mature V2a interneurons. These interneurons are critical for relaying motor signals and represent a promising therapeutic cell population for treating spinal cord injuries. We demonstrated successful enrichment for V2a interneurons in HA-Alg hydrogels containing ECM-derived peptides. Interestingly, both our newly described HA-Alg and established crosslinked HA-HA hydrogels containing IKVAV peptides demonstrated significantly increased neurite length in interneuron enriched cultures compared to hydrogels without peptides. This study demonstrates that the addition of ECM-derived peptides is essential to support the neuronal adhesion and viability required for functional CNS tissue repair. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/729352v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@666b1aorg.highwire.dtl.DTLVardef@17d4borg.highwire.dtl.DTLVardef@1c8ece2org.highwire.dtl.DTLVardef@37fdd5_HPS_FORMAT_FIGEXP M_FIG C_FIG Statement of SignificanceDevelopment of injectable, peptide-modified hyaluronic acid alginate hydrogels to support neuronal maturation for central nervous system tissue repair.

Ovarian tissue cryopreservation and autotransplantation (OTCT) is a crucial fertility preservation strategy for patients facing gonadotoxic cancer treatments, but its clinical success is hampered by ischemic injury and follicle loss following transplantation. This study aimed to enhance OTCT outcomes by employing microporous annealed particle (MAP) hydrogels to promote human ovarian graft revascularization. Unlike non-encapsulated tissue grafts, which exhibited early but transient and disorganized host vascular infiltration followed by regression, tissue grafts encapsulated in MAP hydrogels (OvaMAPs) demonstrated delayed yet organized and stable, long-term revascularization. OvaMAPs had significantly greater mouse CD31+ tissue area and vessel length after 3 and 6 weeks post-transplantation in ovariectomized immunodeficient mice compared to non-encapsulated grafts. By 20 weeks, both groups restored physiological estradiol levels (with OvaMAPs reaching 158 pg/mL) and suppressed follicle-stimulating hormone, confirming integration of the grafts with the hosts hypothalamic-pituitary axes. Notably, OvaMAPs achieved comparable endocrine function restoration with reduced estradiol variability, indicating more consistent graft function. In conclusion, MAP hydrogel encapsulation promoted long-term graft revascularization and vascular stability after OTCT, ultimately supporting consistent endocrine integration with host physiology.

Rapid revascularization is critical to tissue graft survival, as delayed reperfusion drives tissue ischemia and compromises cell viability and graft function. Although bulk hydrogels have been explored for promoting vessel formation, vascularization remains too slow to prevent ischemic injury to grafted tissues, highlighting the need for biomaterial platforms that accelerate graft revascularization and reperfusion. In this study, we present granular hydrogel composites (GHCs), where interstitial space is filled with fibrin and collagen to provide a vasculogenic matrix environment. GHCs supported the assembly of embedded endothelial cells into interconnected, lumenized networks in vitro which anastomosed with host vasculature and were systemically perfused 7 days after implantation. Careful optimization studies revealed that GHCs formed from covalently interlinked, RGD-functionalized microgels of 115 {micro}m diameter best supported vascular network formation in vitro and intravascular blood perfusion in vivo. To test the utility of GHCs for the vascular integration of a demanding and therapeutically relevant parenchymal tissue, GHC-based ovarian tissue grafts were implanted in a murine xenograft model and successfully connected to host vasculature, restoring blood flow to embedded human ovarian tissues within 10 days post-implantation. Notably, endothelial cells seeded within GHCs formed viable vasculature without pre-culture. This work establishes GHCs as a biomaterial platform to rapidly connect parenchymal tissues to host vasculature, with broad translational potential across engineered tissue grafting applications.

Chondral and osteochondral repair strategies are limited by adverse bony changes that occur after injury. Bone resorption can cause entire scaffolds, engineered tissues, or even endogenous repair tissues to subside below the cartilage surface. To address this translational issue, we fabricated poly(D,L-lactide-co-glycolide) (PLGA) microcapsules containing the pro-osteogenic agents triiodothyronine and {beta}-glycerophosphate, and delivered these microcapsules in a large animal model of osteochondral injury to preserve bone structure. We demonstrate that developed microcapsules ruptured in vitro under increasing mechanical loads, and readily sink within a liquid solution, allowing for gravity-based positioning onto the osteochondral surface. In a large animal, these mechano-active microcapsules (MAMCs) were assessed through two different delivery strategies. Intra-articular injection of control MAMCs enabled fluorescent quantification of MAMC rupture and cargo release in a synovial joint setting over time in vivo. This joint-wide injection also confirmed that the MAMCs do not elicit an inflammatory response. In the contralateral hindlimbs, chondral defects were created, MAMCs were locally administered, and nanofracture (Nfx), a clinically utilized method to promote cartilage repair, was performed. The NFx holes enabled marrow-derived stromal cells to enter the defect area and served as repeatable bone injury sites to monitor over time. Animals were evaluated 1 and 2 weeks after injection and surgery. Analysis of injected MAMCs showed that bioactive cargo was released in a controlled fashion over 2 weeks. A bone fluorochrome label injected at the time of surgery displayed maintenance of mineral labeling in the therapeutic group, but resorption in both control groups. Alkaline phosphatase (AP) staining at the osteochondral interface revealed higher AP activity in defects treated with therapeutic MAMCs. Overall, this study establishes a new micro-fluidically generated delivery platform that releases therapeutic factors in an articulating joint, and reduces this to practice in the delivery of therapeutics that preserve bone structure after osteochondral injury.

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.

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.

Osteoporotic bone degeneration involves progressive deterioration of trabecular microarchitecture, yet most scaffold-based bone tissue engineering studies evaluate osteogenesis in structurally favorable architectures that poorly represent compromised bone environments. Here, we establish a degeneration-inspired Voronoi scaffold platform in which point spacing serves as a single tunable architectural parameter to model transitions from dense mechanically integrated to severely deteriorated trabecular-like microenvironments. Increasing point spacing from 1.25 to 2.5 mm progressively reduced scaffold connectivity and stiffness while shifting deformation behavior from distributed load transfer to localized stress concentration, as confirmed by finite element analysis and mechanical testing. Benchmarking against clinically reported HR-pQCT datasets from postmenopausal women demonstrated that the intermediate 1.75 mm point spacing scaffold represents a clinically relevant compromised trabecular-like state, whereas the 2.5 mm scaffold represents a more severely deteriorated architectural condition. These architecture-dependent mechanical and structural transitions directly regulated hMSC behavior, where high point spacing scaffolds reduced cytoskeletal organization, stress fiber density, and osteogenic mineralization, establishing an architecture-associated osteogenic dysfunction regime. Polydopamine (PDA) coating progressively enhanced cytoskeletal organization and mineralization within architecturally compromised scaffolds without altering scaffold geometry. To quantitatively assess biointerface-mediated functional recovery, a Mineralization Rescue Percentage (MRP) framework was introduced, demonstrating up to 43% restoration of architecture-associated mineralization loss following PDA coating. Collectively, this work establishes a clinically contextualized degeneration-to-rescue biomaterials framework that shifts current scaffold design paradigms beyond structurally favorable architectures toward systematic investigation and functional rescue of architecture-associated osteogenic dysfunction within compromised bone-like microenvironments. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/725650v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@26833forg.highwire.dtl.DTLVardef@72b2b7org.highwire.dtl.DTLVardef@333083org.highwire.dtl.DTLVardef@b5f2d1_HPS_FORMAT_FIGEXP M_FIG C_FIG Statement of SignificanceMost scaffold-based bone tissue engineering studies evaluate osteogenesis in structurally favorable architectures that poorly represent compromised bone microenvironments associated with osteoporosis. Here, a clinically contextualized Voronoi scaffold platform is established in which point spacing serves as a single tunable architectural parameter to model transitions from mechanically integrated to structurally deteriorated trabecular-like states. By decoupling architectural and surface biointerface effects, the study demonstrates that architectural deterioration alone can drive cytoskeletal disruption and osteogenic failure. Importantly, polydopamine-mediated surface engineering partially restored cytoskeletal organization and mineralization within architecturally compromised scaffolds without altering bulk geometry. A Mineralization Rescue Percentage (MRP) framework was further introduced to quantitatively assess biointerface-mediated functional recovery within degeneration-inspired scaffold microenvironments.

Osteochondral defects remain a major clinical challenge due to the limited regenerative capacity of cartilage and the complexity of the osteochondral interface. Here, we present a human-derived granular extracellular matrix (gECM) hydrogel platform designed for translational osteochondral repair. Using otherwise discarded human donor tissues, we developed cartilage and bone gECM hydrogels under current good manufacturing practice workflows. These materials are shear-thinning, immediately hold their form, and crosslink under physiological conditions to form stable constructs. Proteomic analysis confirmed that cartilage and bone gECM retain distinct tissue-specific biochemical signatures, while mechanical characterization demonstrated tissue-relevant stiffness, with bone gECM hydrogels exhibiting greater stiffness than cartilage gECM hydrogel. Particle packing density primarily governed viscosity, whereas tissue type contributed strongly to bulk stiffness. Together, these findings establish a scalable, human-derived gECM platform that integrates tissue-specific structural and mechanical cues, and advances a clinically translatable strategy for osteochondral repair.

Neurological disorders are a major cause of death and disability worldwide. The brain's energy metabolism is essential to its proper function, yet the mechanisms driving neuroenergetic dysfunction remain poorly understood. A key challenge is the limited availability of human-relevant models that can reproduce the complexity of brain physiology. An Organ-on-Chip (OoC) system was developed to mimic the neurovascular unit metabolic coupling by incorporating human isogenic iPSC-derived endothelial-like cells, pericyte-like cells, astrocytes, and a cerebral organoid, representing the main cellular components of the NVU. The novel, customized microfluidic platform enables research on neurovascular coupling by interconnecting a blood-brain barrier-on-a-chip model with a 3D brain parenchymal compartment to mimic physiological conditions.

Understanding disease pathology and evaluating emerging therapeutics require in vitro models that accurately recapitulate human tissue environments. However, existing microphysiological systems often compromise either biomimicry or ease-of-use, limiting widespread adoption and scalability. Here, we present lyophilized granular extracellular matrix (gECM) hydrogel wafers as shelf-stable, humanized 2.5D substrates that enable physiologically relevant modeling while simplifying integration into experimental workflows. Derived from decellularized human cartilage and bone, gECM hydrogel wafers retain tissue-specific architecture while introducing microporosity and surface topography through lyophilization. These wafers maintain swelling behavior, structural integrity, and mechanical properties over three months of room-temperature storage, allowing pre-fabrication and on-demand use without loss of function. gECM hydrogel wafers support direct cell seeding without encapsulation and sustain viability and proliferation of human adipose-derived mesenchymal stromal cells over 21 days, with gene expression trends comparable to 3D gECM hydrogels. Furthermore, wafers can be readily integrated into microfluidic systems with in situ hydration and transport of large biomolecules. Together, this platform bridges the gap between conventional 2D culture ease-of-use and 3D biomaterial biomimicry, providing a scalable and physiologically relevant in vitro model approach for high-throughput disease studies and therapeutic screening.

Engineering the human breast in 3D physio-mimetic models is challenging due to its complex multilayered tubular organization, where milk is produced in acini and transported through ductal structures. These functions rely on a highly organized architecture comprising stromal, epithelial, and extracellular matrix compartments. The dysregulation of this architecture perturbs mammary gland homeostasis and promotes the emergence of diverse breast cancer subtypes, from frequent in situ luminal to rarer metastatic basal-like tumors. Despite this knowledge, conventional anti-cancer drug testing still primarily employs high-throughput 3D spheroid models that account for diffusion but lack stromal components, thereby failing to capture stroma-driven treatment resistance. With a unique microfluidic co-extrusion platform, we have developed 3D tubular tissues anchored on a porous and biocompatible alginate shell. Using a one-step protocol, we have bioengineered six relevant ductoid models of healthy and tumoral mammary ducts, most notably a multi-layered model comprising a lumen, mammary epithelium, and stromal compartment made of fibroblasts and matrixes. These new models offer limitless applications in tissue engineering including the characterization of an epithelium and its secretory function, and the identification of the stromal influence on healthy and tumoral mammary gland tissue. Finally, by releasing mechanical constraints, we scale-up the tubular duct model into a mammary assembloid that exhibits branching and budding of acini-like structures from the original duct. We envision that this modular design will broadly impact breast basic and clinical research by opening new experimental avenues toward more physio-mimetic tools through the integration of stromal compartments.

Generating bioengineered cartilage that recapitulates the depth-dependent phenotype, structure, and function of native articular cartilage remains a challenge. While cartilage is rich in aggrecan and type II collagen, proper function depends on depth-dependent protein expression. Superficial zone chondrocytes (SZCs) secrete proteoglycan-4 (PRG4) to lubricate the cartilage surface. Deep zone chondrocytes produce type X collagen (COLX) to support compressive loading and load transfer to subchondral bone. We previously demonstrated that passaged full-thickness chondrocytes (FTCs) and zonal chondrocytes can re-express cartilage and zone-specific markers following scaffold-free three-dimensional (3D) culture in redifferentiation media. However, in the absence of an instructive matrix, cells expressed low levels of zone-specific proteins and exhibited limited depth-dependent organization. We hypothesize that synthetic extracellular matrix with zone-specific microenvironmental cues will guide zonal differentiation. To this end, passaged primary bovine chondrocytes were encapsulated in a soft, hyaluronan (HA)-based, cell-adhesive, and protease-degradable hydrogel established via bioorthogonal tetrazine (Tz) ligation with norbornene (Nb). When supplemented with TGF{beta}3, FTCs deposited aggrecan and type II collagen with minimal type I collagen. Application of interfacial tetrazine ligation with trans-cyclooctene (TCO) during cell culture resulted in matrix stiffening, leading to upregulation of COLX expression. Conversely, SZCs cultured in soft hydrogels exhibited the greatest PRG4 expression. Establishment of a trilayered construct with region-specific stiffness via the diffusion-controlled reaction promoted PRG4 and COLX expression in defined zones. Together, these findings demonstrate that tunable HA-based hydrogels can enhance zone-specific chondrocyte phenotypes and promote the formation of zonally organized cartilage.

Critical sized craniomaxillofacial bone defects do not heal naturally and often exhibit chronic inflammatory responses that restrict regeneration. It is increasingly apparent that biomaterials must facilitate dynamic crosstalk between immune cells, such as macrophages, and osteoprogenitors to resolve inflammation and accelerate regeneration. Here, we evaluate interactions between macrophages in a neutral (M0) or pro-inflammatory (M1) state with mesenchymal stem cells (MSCs) in a basal or licensed state within a mineralized collagen scaffold. We reveal that MSC-macrophage crosstalk influences significant changes in osteoprogenitor cell differentiation and immune cell polarization. Notably, crosstalk between MSCs and macrophages drives an early-stage inflammatory response, which enhances the immunomodulatory activity of MSCs via secretion of IL-6, an effect that is heightened for already licensed MSCs. The presence of macrophages in the co-cultures upregulated osteogenic (ALPL, BMP2, COL1A2, and RUNX2) and angiogenic genes (ANGPT1) in basal MSC groups. Further, MSC-macrophage interactions subsequently drive increased M2-like macrophage polarization as early as 7 days of culture, as indicated by surface marker expression. These findings show that biomaterial scaffolds can be leveraged as mediators of MSC-mediated immunomodulation with an emphasis on achieving early-stage pro-inflammatory phenotypes that drive subsequent macrophage polarization and markers of increased regenerative potency.