Tissue Engineering Part A

Successful cartilage repair remains one of the most significant challenges in the musculoskeletal field. Microfracture (MFx), a form of marrow stimulation, remains the predominant repair technique, but it exhibits routine failure due to inadequate defect fill and inferior fibrotic tissue formation. Whereas current strategies focus on augmenting MFx with scaffolds and bioactive factors, the potential to target the MFx clot itself and use the capabilities of this dynamic environment to guide MFx repair remains largely unexplored. We verified that MFx contraction and fibrosis hinder repair success in minipigs and become evident as early as one week in multiple animal models. Therefore, our objective was to investigate and direct microenvironmental interactions in the MFx clot to promote volumetric maintenance and reprogram cells from a fibrotic to more chondrogenic phenotype. Extracellular control of cell-environment interactions, through fibrinogen augmentation or anti-fibrinolytic treatment, limited contraction but had no effect on or even exacerbated the fibrotic susceptibility of marrow-derived cells (MDCs). Intracellular control of microenvironmental interactions, through modulation of the Rho-ROCK pathway, drove TGF-{beta}3 activity of MDCs along a "chondro-fibro axis". In particular, treatment with the ROCK inhibitor Fasudil drove TGF-{beta}3-treated cells away from a myofibroblast phenotype and towards chondrogenesis. Short-term Fasudil treatment prevented TGF-{beta}3-driven macroscale clot contraction and enhanced cartilage-specific matrix deposition in vitro. In a pilot rat study, this combination treatment improved GAG deposition and better protected surrounding cartilage. These findings suggest that Rho-ROCK modulates TGF-{beta} signaling along this chondro-fibro axis and its precise control could be the key to promoting precise and volumetric cartilage repair through microenvironmental interactions.

Cell therapies involving human adipose-derived stromal cells (hASCs) have shown promise for a range of clinical applications due to their ability to stimulate angiogenesis and dampen inflammation via paracrine mechanisms. However, a major barrier to the successful clinical translation of hASC-based therapies is that standard culture methods for expansion on rigid 2D tissue-culture polystyrene under static conditions diminish the pro-regenerative functionality of the cells. To address these limitations, the current project focused on the development of an in vitro bioreactor system for preconditioning hASCs to augment their capacity to stimulate regeneration through paracrine mechanisms. Specifically, the combined effects of decellularized adipose tissue (DAT) coatings, shear-stress stimulation, and varying oxygen tensions on hASC expansion and paracrine factor secretion were assessed. Additional studies were performed to characterize the effects of stimulating hASCs within the rocking bioreactor system using the pro-inflammatory cytokines IFN-{gamma} and TNF-. Expansion in the bioreactor under all conditions supported hASC growth with no observable morphological differences. However, dynamic culture on DAT coatings enhanced intracellular indoleamine 2,3-dioxygenase (IDO) expression in hASCs cultured under 20% O2. Moreover, culturing under dynamic conditions and/or on DAT coatings significantly increased secretion of the pro-angiogenic factors VEGF, HGF, and angiogenin. When pro-inflammatory cytokine priming was introduced, the expression of all tested paracrine factors was enhanced, particularly the immunomodulatory factors IL-6, IL-8 and MCP-1. Overall, a novel bioreactor system was developed for hASC expansion and preconditioning, demonstrating that the cell microenvironment can be tuned to modulate hASC paracrine factor secretion.

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.

Collagen, a major component of the extracellular matrix, regulates cellular behaviors, such as adhesion, differentiation, and angiogenesis. These functions are mediated by interactions between specific amino acid motifs within the collagen triple-helical structure and collagen-binding biomolecules. These include cell-surface receptors, such as integrins, discoidin domain receptors (DDRs), and syndecans, a family of transmembrane heparan sulfate proteoglycans (HSPGs). Signals mediated by these receptors are integrated to regulate cell fate. However, native collagen simultaneously presents multiple receptor-binding motifs, making it difficult to isolate receptor-specific functions and to evaluate receptor crosstalk. Here, we introduce a composition-controlled artificial collagen matrix platform that enables independent tuning of multiple receptor-binding motifs within a constant triple-helical scaffold. This material was produced by disulfide crosslinking of chemically synthesized collagen-like triple-helical peptides, each bearing a single defined receptor-binding sequence. By varying the mixing ratios of these peptides before crosslinking, we systematically controlled the composition of receptor-binding motifs within the matrices. We applied this platform to nerve growth factor-dependent neuronal differentiation of PC12 cells, a process supported by collagen. Matrices containing only integrin-binding sequences were sufficient to support this differentiation. Incorporation of an HSPG-binding sequence had little additional effect, whereas incorporation of a DDR-binding sequence suppressed integrin-mediated differentiation and coincided with DDR phosphorylation. These results reveal opposing roles of collagen-binding integrins and DDRs in regulating PC12 cell differentiation. Composition-controlled artificial collagen provides a versatile matrix platform for dissecting functional crosstalk among collagen receptors.

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.

Tympanic membrane perforations remain a common clinical problem, and although surgical intervention through tympanoplasty achieves high success rates, it is associated with donor-site morbidity, surgical complexity and limited restoration of the native radial and circumferential collagen architecture. In this study, 3D extrusion printing was utilized to create an active scaffold and attempt to promote collagen organization through shear-mediated structural alignment. An alginate-carboxymethyl cellulose (CMC) hydrogel with bovine SIS-dECM was prepared and investigated for its suitability as a bioink alternative to tympanoplasty grafts. The physiochemical, rheological and printability characteristics of the hydrogel were assessed. Successful decellularization was confirmed by histological analysis. The incorporation of the SIS-dECM into the hydrogel led to increased swelling, lower apparent viscosity, yield stress and flow stress while maintaining favourable printability and filament stability. Polarized optical microscopy was also used to study the influence of printing speed on the alignment of collagen to mimic the native tympanic membrane radial collagen architecture. Compared with the cast controls, the printed samples presented stronger birefringence signals. Biological evaluation demonstrated that the 15% dECM hydrogel exhibited the highest live cell area percentage and live/dead ratio after 48 h. In addition, the chick chorioallantoic membrane assay demonstrated that the dECM-containing hydrogels improved vascular density. The findings establish a printable, biologically active dECM bioink capable of generating bulk collagen organization through extrusion printing as a platform for tympanic membrane regeneration.

Decellularized extracellular matrices (ECMs) are widely used in regenerative medicine, yet current evaluation criteria prioritize cellular removal rather than preservation of the ECM characteristics that govern tissue behavior. Here, we demonstrate that efficient decellularization is achieved across a broad range of chemical conditions, whereas preservation of structurally and biologically relevant ECM components is confined to narrow, tissue-specific windows defined by coupled detergent interactions. Quantitative proteomics revealed that intrinsic ECM composition is strongly associated with tissue-specific susceptibility to decellularization-induced damage and provided molecular context for the distinct preservation responses between tissues. Optimized matrices retained major structural ECM components and supported tissue-specific cellular organization and cell-mediated mechanical reinforcement following cellular repopulation despite uniformly low residual DNA across protocols. Together, these findings support a shift in decellularization quality assessment from DNA-based evaluation toward preservation of biologically relevant ECM and establish a composition-driven strategy for the rational design of regenerative biomaterials with tissue-relevant biological and mechanical properties.

Sensory and motor neurons differ significantly in their morphology, structural organization, functional properties, and mode of action. However, despite this heterogeneity, many in vitro studies focus only on a single neuronal subtype, mainly being sensory neurons, limiting the translational potential and relevance of these studies for the evaluation of therapeutic options for spinal cord injury. In this study, we investigate the differentiation, maturation, and neuronal outgrowth of human induced pluripotent stem cell (iPSC)-derived motor and sensory neurospheres using polyethylene glycol (PEG)-microgels with various bioactive coatings. Our results show subtype-specific responses to the PEG-microgel scaffolds, with respect to motor and sensory neurosphere morphology and size. Furthermore, we compare the formation of the PEG-microgel/scaffolds when starting from iPSCs-derived precursor neuron spheres versus undifferentiated iPSCs. We observe notable differences in structural organization, maturity, and neuronal outgrowth between the two approaches, as well as between motor and sensory neurospheres. Together, these results underline the importance of studying motor and sensory neurons separately and highlight the need for a controlled, tunable culture platform to assess the impact of the microenvironment and to improve the physiological relevance of in vitro platforms for neuron-based research.

Endothelial cells (ECs) derived from human induced pluripotent stem cells (hiPSCs) are increasingly used to model vascular diseases and test therapeutic strategies. However, the efficiency and reproducibility of differentiation can vary depending on the culture medium and its supplemented factors and stages. Here, we directly compared two defined media, APEL and BPEL, for iPSC-to-ECs differentiation. iPSCs were differentiated over 10 days with sequential growth factor induction, followed by magnetic-activated cell sorting or flow cytometry for CD31+ cells. Both media produced ECs with similar morphology and marker expression, including CD31 and VE-cadherin. Functional assays demonstrated comparable tube formation, indicating equivalent endothelial functionality. Cost analysis indicated that APEL had a higher total reagent cost but generated a higher total cell yield, resulting in a comparable cost per 10 total cells, whereas BPEL was more cost-efficient for producing CD31/VE-cadherin endothelial-specific cells. Our results suggest that APEL and BPEL media are equally effective for generating iPSC-derived ECs, providing flexibility in method selection for vascular disease modeling and drug discovery applications.

Engineered heart tissues (EHTs) represent an innovative platform enabling physiologically relevant in vitro evaluation of drug-induced cardiac responses. While functional characterization remains central to EHTs, molecular profiling is increasingly used to elucidate mechanisms underlying drug-induced phenotypes. Proteomics provides broad molecular characterization of drug responses at the protein level, yet the complexity, heterogeneity, and high dimensionality of proteomics datasets challenge conventional statistical approaches, which are not designed for cross-modal integration and streamlined multi-omics analysis. In this study, we developed an innovative framework based on topological data analysis (TDA) for the integration of large proteomics profiles and functional readouts to investigate system-level responses to drugs with opposing inotropic effects, epinephrine and doxorubicin. Samples were organized into a topological connectivity network according to multimodal similarity enabling simultaneous exploration of treatments, cardiac function and proteome alterations. Highly correlated features were then used for pathway enrichment analysis, which revealed strong similarities between the enrichment profiles associated with contractile force and epinephrine. These findings are consistent with the positive inotropic effect of epinephrine, whereas doxorubicin exhibited an opposing enrichment profile. Energy homeostasis, mitochondrial translation and proteostasis emerged as the major cellular processes displaying opposite associations with the two inotropic drugs, highlighting a link between cardiac contractility and perturbations in these processes. In conclusion, our TDA-based framework successfully integrated functional and proteomic data to uncover treatment-specific remodeling in EHTs, offering a modular and scalable approach that could be adapted to other in vitro organ models for systems-level mechanistic studies and next-generation drug development.

Disruption of the wound healing cascade can result in pathological outcomes, including fibrosis due to myofibroblast-mediated contraction and collagen deposition. Despite the clinical significance, effective treatments for fibrosis remain limited as current therapies often show inconsistent efficacy, adverse effects, and patient discomfort. Combinatorial therapeutic strategies integrating biomaterial scaffolds with gene delivery have shown promise in regenerative healing. MicroRNAs (miRNAs) are key regulators of fibrotic signalling in cells, including fibroblasts and myofibroblasts. Specifically, miRNA-29b is notable for downregulating pro-fibrotic genes, including collagen type I, reducing ECM accumulation, and limiting fibroblast/myofibroblast overactivation. In this context, the present work develops a collagen-GAG (CG) scaffold platform for delivery of miRNA-29b complexed GET nanoparticles to inhibit fibrosis. Initially, bioinformatic analysis of miRNA-29b validated its involvement in ECM-associated pathways and processes, followed by successful nanoparticle internationalisation in primary dermal fibroblasts. The anti-fibrotic efficacy of the optimised miRNA-29b nanoparticles was subsequently demonstrated by significant reductions in collagen deposition and -SMA expression, both key indicators of myofibroblast differentiation and fibrosis. The optimised miRNA-29b formulation was then incorporated into 3D porous collagen-GAG (CG) scaffolds, which modulated fibrotic gene expression while preserving scaffold structure conducive to fibroblast/myofibroblast infiltration and proliferation. Finally, functional outcomes of seeded TGF-{beta}-stimulated fibroblasts, including reduced matrix contraction, -SMA expression, and ECM deposition, were comparable to those observed in non-fibrotic conditions, thereby confirming the therapeutic potential of scaffold-mediated miRNA-29b delivery. Together, these findings demonstrate that scaffold-mediated miRNA-29b delivery represents a promising anti-fibrotic strategy for wound healing by mitigating myofibroblast activation, limiting matrix contraction, and preventing pathological ECM accumulation.

The foreign body response (FBR) is an immune mediated event that occurs with every material implant. The extent of the fibrosis is dependent on many factors including the biomaterial design, tissue location, and host factors such as age, sex, ancestry, diet. There are known clinical outcomes of implants dependent on age and sex, including increased fibrosis and implant failure in aged and female patients. As the population ages, there is a growing need to understand how aging affects the FBR, and how preclinical models can capture this to guide biomaterial design. Here, we investigated how chronic fibrosis in a murine model of the FBR is altered by two biological factors: age and sex. We investigated changes in fibrosis using a volumetric muscle loss (VML) injury model coupled with polycaprolactone (PCL) or polyethylene (PE) microparticle implants. Fibrosis was quantified through gene expression, microscopic analysis of histologic sections, and the corresponding immune response measured via gene expression and flow cytometry data. We found gene expression differences with immune pathways enriched in female mice, and microscopy revealed collagen birefringence area increased in young male mice. Both the innate and adaptive immune response were altered by age and sex via T cell and macrophage phenotype, and the effects of aging differed between sexes. These results reveal both variables contribute to discrepant outcomes in both fibrosis and the local immune response to synthetic material implants. This demonstrates a clear need to understand and account for the influence of biological factors in biomaterial design.

Embedded extrusion printing can process collagen-rich bioinks, but their low viscosity and slow fibrillogenesis compromise print fidelity and post-deposition stability. Here, we developed a collagen fibril-inducing support bath (FIB) that combines mechanical support for embedded printing with biochemical induction of collagen assembly. Microfibrillated or nanofibrillated cellulose was incorporated into a fibril-inducing buffer, and formulations were screened at 37 degrees Celsius for rheological behaviour and optical transparency. The selected FIB was evaluated by printing 1% and 5% (w/v) articular cartilage-derived extracellular matrix (ECM) inks at 10-20 mm/s and compared with a cellulose-only control bath. FIB exhibited yield-stress, shear-thinning and rapid recovery behaviour that supported reproducible filament deposition. Unlike the control bath, FIB enabled intact construct retrieval following stabilisation and promoted the formation of fibrillar collagen within the printed strands. Scanning electron microscopy revealed D-banded collagen fibrils preferentially oriented along the deposition direction, with dominant orientation peaks within +/- 10-15 degrees. The platform supported the fabrication of 15 x 15 x 1.5 mm sheets and 6 x 6 x 6 mm scaffolds whose macroscopic dimensions were retained after processing. Constructs produced from 5% ECM inks exhibited approximately fourfold higher ramp and relaxation moduli than those produced from 1% ECM inks. Extracts from both formulations caused no detectable reduction in cell metabolic activity after 24 h or 72 h. Mesenchymal stem/stromal cells (MSCs) seeded onto printed sheets became markedly elongated and aligned by day 3, with approximately 80% of cells having an aspect ratio exceeding 1.5, significantly greater than cells seeded onto casted ECM controls, with a mean deviation of ~9 degrees from the filament print direction. These findings establish FIB as a bioactive support bath that couples embedded printability with collagen fibrillogenesis, enabling recoverable collagen-rich constructs with aligned fibrillar architecture that directs early cellular organisation.

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.

Introduction: Advanced biomaterials for regenerative medicine are increasingly expected to combine multifunctionality and compatibility with tissue-specific cellular processes. In this context, hydroxyapatite-based platforms modified through ionic substitution represent promising candidates, as they may integrate structural similarity to bone mineral with additional biological functionality and luminescent properties, enabling diagnostic applications and real-time monitoring. In this study, we evaluated whether silicate-phosphate substituted calcium hydroxyapatite Ca10(PO4)6-x(SiO4)x(OH)2 (where x = 1.5) co-doped with lithium(I), europium(III), and gadolinium(III) ions (Si-HAp-LEG) affects the osteogenic, chondrogenic, and adipogenic differentiation potential of human bone marrow stromal/stem cells (BMSCs). Methods: Human BMSCs were cultured under lineage-specific differentiation conditions in the presence of undoped silicate-substituted phosphate hydroxyapatite (abbr. as Si-HAp), which served as a control, and two distinct Si-HAp-LEG formulations differing in gadolinium(III) (Gd3+) as well as lithium (Li+) and europium(III) (Eu3+) ion concentrations: Si-HAp-LEG-221 (1 mol% Gd3+ ion) and Si-HAp-LEG-222 (2 mol% Gd3+ ion). Differentiation-associated phenotypic outcomes, including extracellular matrix formation and lipid accumulation, were evaluated using Safranin O, Alizarin Red, and Oil Red O staining. In parallel, biomaterial-induced molecular responses were characterized at the transcriptomic and protein levels using RT-qPCR for selected coding and non-coding RNAs and Western blot analysis for representative lineage-associated proteins. Results: Histochemical evaluation confirmed that, across all tested biomaterial groups, BMSCs retained the ability to form mineralized calcium deposits, proteoglycan-rich extracellular matrix, and intracellular lipid accumulation under osteogenic, chondrogenic, and adipogenic conditions, respectively. Quantitative staining analysis revealed no significant Si-HAp-LEG-dependent enhancement of terminal differentiation outcomes compared with undoped Si-HAp. In turn, the molecular response differed between biomaterials modifications. Si-HAp-LEG-222 induced the most prominent changes in transcriptional and post-transcriptional regulators, particularly within BMP/SMAD-associated pathways under osteogenic and chondrogenic conditions, underlying a potential link between gadolinium concentration and osteogenic lineage commitment. However, these transcriptomic responses were not mirrored by consistent changes at the protein level. The results suggest that silicate-phosphate substituted hydroxyapatite co-doped with Li+, Eu3+, and Gd3+ ions primarily affects the early regulatory pathways associated with BMSCs differentiation rather than enhancing their terminal maturation. Discussion: In conclusion, the collective data indicate that Li+, Eu3+, and Gd3+ ions LEG co-doping broadens the multifunctional potential of Si-HAp by introducing imaging-related properties while preserving its underlying pro-regenerative character. Li+, Eu3+, and Gd3+ ions co-doped LEG-substituted Si-HAp may therefore be considered a compatible biomaterial platform that maintains BMSC cellular plasticity and supports balanced, differentiation-dependent modulation of lineage-associated molecular responses.

Vascularization, which is critical for most engineered tissue constructs, is dependent on interactions between endothelial cells and spatiotemporally presented biochemical and biophysical cues. Yet, most hydrogels define these cues at the time of fabrication, thus precluding adjustments to actively drive vascular formation. We developed acoustically responsive scaffolds (ARSs) that use focused ultrasound to trigger growth factor release and localized matrix remodeling within fibrin hydrogels. ARSs were formed by incorporating phase-shift emulsions containing basic fibroblast growth factor (bFGF) along with perfluorohexane (C6) or perfluorooctane (C8). Upon ultrasound exposure, stable bubbles were generated in C6-ARSs that locally compacted the matrix and increased macroscale stiffness. Comparatively, in C8-ARSs, ultrasound generated macropores without impacting viscoelastic properties. Ultrasound increased bFGF release from both ARS types, which enhanced in vitro and in vivo vasculogenic assembly in ARSs with co-encapsulated endothelial cells and fibroblasts. Our data also show that ultrasound-driven matrix remodeling without bFGF release increased endothelial sprouting. In C6-ARSs, elevated levels of F-actin were observed in both cell types adjacent to bubbles as well as increased YAP intensity and nuclear asymmetry. Together, these results establish ARSs as reconfigurable hydrogels that couple on-demand release of biochemical cues with programmable matrix restructuring to direct three-dimensional microvascular assembly.

BackgroundOwing to their potent immunomodulatory properties, mesenchymal stromal cell (MSC)-derived small extracellular vesicles (EVs) have emerged as promising neuroprotective treatments for ischemic stroke. Preclinical studies using MSC-EVs have mainly been performed in young, otherwise healthy rodents. Stroke patients frequently carry vascular risk factors and comorbidities. We herein investigated whether MSC-EVs retain neuroprotective activity in hyperlipidemic mice on cholesterol-rich Western diet. MethodsMale C57BL/6J mice were exposed to regular normal diet or Western diet for 6 weeks. At the age of 9-10 weeks, mice were exposed to transient intraluminal middle cerebral artery occlusion (MCAO). Vehicle or MSC-EVs (2x106 or 6x106 cell equivalents) were intravenously administered immediately after reperfusion, and vehicle or rosuvastatin (5 mg/kg/day) were intraperitoneally applied starting immediately after or seven days before MCAO. Neurological deficits, ischemic injury, and immune responses were evaluated up to 72 hours post-ischemia. To investigate the hyperlipidemia-associated immune dysregulation, mice received DNase-I before or immediately after MCAO. In defined subgroups, monocytes/ macrophages or neutrophils were additionally depleted by clodronate liposomes or anti-Ly6G antibodies, respectively. ResultsIn contrast to normolipidemic control mice, MSC-EVs failed to induce post-ischemic neuroprotection in hyperlipidemic mice. Neither MSC-EV dose escalation nor rosuvastatin co-treatment restored the therapeutic efficacy of MSC-EVs. Hyperlipidemia induced systemic innate immune dysregulation characterized by reduced monocyte/ macrophage activation, increased neutrophil activation, and elevated circulating cell-free DNA. DNase-I treatment before, but not after MCAO reversed these immune abnormalities and restored neuroprotection by MSC-EVs, decreasing neurological deficits, infarct volume and brain edema. Depletion of either monocytes/ macrophages or neutrophils abolished the neuroprotective effects of MSC-EVs in DNase-I-pretreated hyperlipidemic mice. ConclusionsImmune dysregulation abolishes MSC-EV-induced neuroprotection after ischemic stroke in hyperlipidemic mice. DNase-I priming restores MSC-EV responsiveness through mechanisms critically involving monocyte/ macrophage and neutrophil rebalancing. Our data highlight the host immune status as determinant of EV therapeutic efficacy.

The inflammatory foreign body response that follows cochlear implantation produces intracochlear fibrosis, neo-ossification, and elevated electrode impedances that can compromise implant performance. Dexamethasone-eluting cochlear implants reduce this response, but the durability of their anti-inflammatory effect over long implantation intervals has not been established. Using a murine model of chronic cochlear implantation in CX3CR1+/eGFP Thy1+/eYFP dual-reporter mice, we compared dexamethasone-eluting and standard mouse cochlear implants at 224 and 336 days post-implantation. Density of CX3CR1+ macrophages, MHCII+CX3CR1+ antigen-presenting macrophages, -SMA+ fibrosis, and neo-ossification were quantified in the scala tympani, Rosenthal canal, and lateral wall of the basal turn. Standard implants produced persistent macrophage and antigen-presenting macrophage infiltration, accompanied by an -SMA+ fibrotic response and neo-ossification. Dexamethasone-eluting implants suppressed macrophage infiltration in all three regions out to 336 days and reduced fibrosis at 224 days. In the subset of cochleae with electrode array translocation, dexamethasone-eluting implants attenuated macrophage infiltration and confined the fibrotic and osseous response to the site of translocation, whereas standard implants produced a widespread response. A reduction in immune cell density was also observed in the contralateral, unimplanted cochleae of animals implanted with dexamethasone-eluting implants, suggesting a wider component to the drug's effect. Dexamethasone-eluting cochlear implants therefore provide sustained, long-term suppression of the cochlear foreign body response in mice, supporting their continued translation toward clinical application. This effect was associated with continued low-level dexamethasone elution out to 336 days post-implantation; further work is needed to assess the durability of this effect at the conclusion of drug elution.

Neurodegenerative diseases are increasingly associated with vascular dysfunction beyond progressive neuronal degeneration, yet how vascular pathology contributes to disease progression remains poorly understood, largely due to the lack of a neurodegenerative disease model capable of capturing neuronal pathology alongside associated vascular dysfunction. Here, we developed a microengineered 3D vascularized brain tissue model that integrates neurospheroids with a self-assembled, perfusable vascular network to recapitulate key features of the neurovascular interface. Using this model, we investigated the vascular contribution to Parkinson's disease pathology by introducing -synuclein preformed fibrils into the engineered vasculature. Intravascular -syn fibril exposure induced endothelial barrier disruption, vascular leakage, inflammation, and vascular regression. Notably, this vascular insult was accompanied by intraneuronal -synuclein aggregation within neurospheroids, suggesting that vascular dysfunction may facilitate the exposure of neural tissue to pathogenic -synuclein. Our neurodegenerative disease modeling approach establishes a versatile and tractable platform for investigating vascular contributions to neurodegenerative disease progression.

Pancreatic beta cells exhibit marked resistance to proliferation, posing a barrier to therapeutic strategies aimed at restoring beta-cell mass in diabetes. Here, we present a transient, non-integrative approach to stimulate beta-cell proliferation using in vitro transcribed (IVT) mRNAs encoding cell cycle regulators. In rodent beta cells and human-beta cell derived EndoC-BH5 cells, chemically modified IVT mRNAs activated cell cycle entry and subsequent mitosis. A single dose of cyclin D1 and CDK4 IVT mRNAs nearly doubled the number of rat beta cells. However, achieving cell division in human beta cells required co-delivery of MYC IVT mRNA. The mitogenic response of beta cells peaked within 36-60 hours, and declined thereafter, reflecting the transient nature of IVT mRNA. Transcriptomic profiling revealed temporary activation of proliferative pathways and reversible downregulation of beta-cell maturation markers. Importantly, we detected no evidence of sustained proliferation. Our findings demonstrate that mRNA-based delivery of cell cycle regulators can overcome the intrinsic cell cycle block in beta cells and may provide a controllable approach for beta-cell regeneration.