Acta Biomaterialia

The human spine is a complex, coordinated biomechanical system. Physiologically, its tissues are also highly interdependent in terms of function and viability. The interaction between mechanical stress and biological/biochemical activity over time constitutes a key driver of spinal degeneration. Research to date providing mechanistic insights into this process has focused on individual components (vertebra and disc tissue analogues), in isolation or as basic functional units. However, many observations from individual units will not translate to whole spine behaviour. The intricate complexity of the spine requires novel experimental models (synthetic and biotic), which must consider the spine at an organ level and adopt an integrative approach that can capture the dynamics which govern its function. Here, we report the development of a biomimetic spinal model prototype, amenable to cellular integration, which is miniaturised to the in vitro scale to provide a controlled environment and testbed for axial biological mechanics. The research presented here encompasses more than a decade of systematic investigations during which the gradual emergence of key manufacturing innovations progressively enabled addressing an exceptionally complex bioengineering challenge - organotypic spine engineering. The model comprises the full anatomical range of spinal vertebrae/bones (C1 to Sacrum & Coccyx), reproduced using bioceramic materials, assembled in sequence into a relevant columnar architecture and mechanically connected end-to-end by biochemically active interfaces. A range of assessments examining anatomical design, material behaviour and manufacturing processes is presented. The work explores concepts such as longitudinal mechanobiology and multi-segment coupling as well as manufacturing strategies using autonomous materials and instrumentation. This prototype introduces for the first time columnar level behaviour and the ability to study time dependent adaptations. This model is important because it can support tissue maturation, evolving mechanical properties and adaptive behaviour and it represents an intermediate step between isolated skeletal tissue models and future organ-level spinal constructs.

Producing bone and cartilage in a controlled and localized manner remains a significant challenge in regenerative medicine. This study investigated the ability of keratin- and polyethylene glycol (PEG)-based degradable hydrogels to deliver bone morphogenetic protein 2 (BMP-2) and leukocyte cell-derived chemotaxin 1 (LECT-1; also known as chondromodulin-1) intramuscularly to induce ectopic tissue formation. Adult male CD-1 mice received intramuscular implants of keratin-PEG gels containing a fixed dose of BMP-2 and increasing amounts of LECT-1. After two weeks, implants and surrounding muscle were analyzed using computed tomography (CT) and histology. The results showed that BMP-2 is necessary for forming new bone and cartilage, whereas LECT-1 alone appeared to trigger muscle dedifferentiation without ossification or chondrogenesis. Co-delivery of BMP-2 and LECT-1 enhanced bone and cartilage formation in a dose-dependent manner: higher LECT-1 doses led to proportionally more ectopic cartilage (linear correlation, r2 {approx} 90%), while bone formation peaked at the third LECT-1 dose at approximately twice the volume of the BMP-2-only group. These findings indicate that muscle-resident cells may be capable of reverting and switching to mesenchymal lineages, recapitulating endochondral ossification. The platform offers a promising strategy for growing bone and cartilage autografts within skeletal muscle bundles.

Control over network dynamics at different length scales is a feature of natural materials challenging to replicate in synthetic hydrogels. Hydrogel viscoelasticity is commonly controlled by tuning the kinetics of reversible crosslinks; however, this strategy inherently links the resulting macroscale and nanoscale dynamics of the individual network components. Taking inspiration from biological materials that feature lipids as structural elements, we introduce Lipid Network Crosslinked (LINC) hydrogels that exploit the mobility of individual lipids within self-assembled liposomes as covalent, network-crosslinking points. These mobile, covalent crosslinks increase hydrogel stress relaxation rates over 20-fold compared to polymer-only hydrogels with equivalent crosslinking chemistries and stiffnesses. We demonstrate that liposome design parameters, including degree of surface functionalization and tail saturation, provide a means to independently control the macroscale storage moduli and stress relaxation behavior. Finally, as an application where control over network dynamics at different length scales is critical, we placed cell-adhesive ligands onto more mobile or less mobile network elements. Human neural progenitor cells cultured within LINC hydrogels of identical macroscale viscoelasticity significantly altered their phenotype in response to nanoscale ligand dynamics. These results establish LINC hydrogels as biomimetic materials that leverage nanoscale lipid mobility within a macroscale polymeric network to control dynamics at multiple length scales.

Muscle satellite cells (SCs) regenerate skeletal muscle, but their regenerative capacity declines with age, in part due to extracellular matrix (ECM) remodeling and aberrant fibroblast activation within the SC niche. In regenerating young mouse muscle, fibronectin remodeling is transient, whereas in aged mouse muscle, fibronectin remodeling is prolonged and disorganized. Fibroblasts in aged mice are activated, increasing fibronectin deposition and expressing elevated -smooth muscle actin (SMA), which negatively influence SC fate. We develop a viscoelastic hydrogel co-encapsulation system, enabling three-dimensional co-culture of intact myofibers with primary fibroblasts. Using this 3D co-culture system, we show that fibroblasts from young mice support SC quiescence and self-renewal, whereas fibroblasts from aged mice aberrantly activate SCs and promote their differentiation on myofibers isolated from either young or aged mice. Knocking down fibronectin (Fn1) in fibroblasts from aged mice partially restores SC function, promoting quiescence and limiting differentiation. Using a novel 3D hydrogel co-culture system, we demonstrate that fibroblast-deposited fibronectin is a key age-associated regulator negatively affecting SC fate within the SC niche of aged mice.

Silk proteins, including sericin and fibroin, are natural biopolymers with broad applications in tissue engineering where angiogenesis plays an essential role. However, the pro-angiogenic effects of silk proteins with varying molecular weights (MWs) remain poorly understood. Here, silk proteins with MW distributions at 40-180 kDa or less than 25 kDa were obtained through alkaline hydrolysis to evaluate their effects on angiogenesis. Structurally, reducing MW induced a conformational transition in silk proteins, accompanied by a striking morphological shift in sericin from nanofibers to nanoparticles. Functionally, high-MW sericin (SSH) suppressed, whereas low-MW sericin (SSL) and both high- and low-MW silk fibroin (SFH/SFL) directly promoted endothelial angiogenic activity. Transcriptomic analysis revealed that angiogenesis-related genes such as Id1 and Smad6/9 may underlie the angiostatic effects of SSH. Notably, both SSH and SSL enhanced angiogenesis indirectly via macrophages; however, SSH induced mixed M1/M2-like polarization, while SSL preferentially drove an M2-like phenotype. In a subcutaneous implantation model, SSH promoted angiogenesis but yielded vessels with weak integrity and increased fibrosis, whereas SSL enhanced angiogenesis with improved vascular maturity and reduced fibrotic response. These findings elucidate how the MWs of silk proteins shape angiogenic behavior and highlight the importance of MW tailoring for optimized tissue engineering applications.

Granular extracellular matrix (gECM)-based biomaterials commonly contain polymer components to improve scaffold cohesion and handling during fabrication and use. However, these polymer hydrogel components may dilute ECM content and increase fabrication and regulatory complexity. This study evaluated whether particle-only gECM wafers could serve as a simplified alternative to hydrogel-based gECM scaffolds while maintaining structural, mechanical, and biological performance. Decellularized human cartilage and skin tissues were processed and fabricated into three scaffold formats: gECM hydrogels, freeze-dried gECM hydrogel wafers, and freeze-dried particle-only gECM wafers. Across fabrication methods, scaffold swelling, volume fraction, and stiffness were strongly influenced by both tissue type and fabrication approach. gECM hydrogels exhibited the greatest swelling and lowest stiffness, while gECM wafers displayed higher volume fractions and greater mechanical stiffness. Notably, gECM particle-only wafers achieved performance comparable to gECM hydrogel wafers despite the absence of a secondary polymer network. Particle-only wafers also maintained swelling behavior and structural properties over 3 months of dry storage at room temperature, with only modest decreases in stiffness. In vitro studies showed sustained cell viability over 14 days on particle-only wafers, with chondrocytes infiltrating cartilage wafers and fibroblasts remaining primarily surface-localized on skin wafers. In addition, particle-only wafers remained cohesive during implantation into a bovine cartilage defect model. These findings demonstrate that particle-only gECM wafers can achieve structural integrity, mechanical performance, and cytocompatibility without the need for an additional polymer network, highlighting a simplified and ECM-rich biomaterial platform. By eliminating polymer carriers and enabling dry storage with preserved function, this approach supports the development of off-the-shelf, translationally accessible gECM particle-only wafers for tissue engineering applications.

ABSTRACTThe repair of large bone defects remains a major clinical challenge, in part due to inadequate vascularization and poor integration of graft materials. Tissue engineering strategies that recapitulate the developmental process of endochondral ossification, whereby a cartilage template remodels into bone, have shown significant potential in pre-clinical models of large bone defect healing. However, successfully scaling these approaches to clinically relevant sizes will require the development of strategies to support the rapid vascularization of the graft following implantation in vivo. Here, mechanically reinforced templates were first fabricated by integrating hypertrophic cartilage microtissues derived from human mesenchymal stem/stromal cells (MSCs) within an osteoconductive 3D-printed polycaprolactone (PCL) framework coated with nano-hydroxyapatite (nanoHA). In vitro the cartilage microtissues fused and generated an extracellular matrix rich in sulphated glycosaminoglycans and collagen. To prevascularize these constructs, vascular microtissues derived from a co-culture of endothelial cells and MSCs were incorporated into a central channel within the construct, which generated a microvascular network within the graft in vitro. Following subcutaneous implantation, hypertrophic cartilage templates with ( vascular-channel group) and without ( empty-channel group) this central vascularized channel supported endochondral bone formation. Quantitative microCT and histological analyses revealed significantly greater remaining bone in the empty-channel group, whereas the vascular-channel group supported enhanced vascularization and remodeling of the graft in vivo. These findings support the continued development and testing of a modular biofabrication strategy that combine self-organizing hypertrophic cartilage and vascular microtissues with osteoconductive 3D-printed architectures to generate scalable, prevascularised hypertrophic cartilage templates for endochondral bone repair. Key-words: spheroids, microtissues, hypertrophic cartilage, vascularization, endochondral ossification, bone tissue engineering.

Biomaterial scaffolds for repairing traumatic muscle injuries require restoration of both the anisotropic architecture and basement membrane extracellular matrix cues critical to normal muscle function. To address this need, we establish a collagen-glycosaminoglycan (CG) scaffold platform pairing an aligned pore microstructure, produced via directional freeze-drying, with basement membrane protein functionalization via carbodiimide crosslinking. Laminin and/or collagen IV are successfully tethered and retained within CG scaffolds over 7 days without significantly altering pore size or alignment, confirming stable protein functionalization and preservation of scaffold architecture. Human muscle progenitor cells show excellent viability and metabolic activity in all scaffold groups, with collagen IV functionalization significantly enhancing myotube number and fusion index. Toward establishing scaffold compatibility with non-myogenic support cells, we show that neural stem cells remain viable and metabolically active across all scaffold conditions. Overall, these findings highlight the combination of aligned scaffold architecture and collagen IV functionalization as potentially impactful for skeletal muscle tissue engineering.

Introduction: Critical-sized bone defects cannot heal spontaneously, requiring additional, often burdensome, treatment. Thus, various synthetic substitute materials have been investigated regarding their treatment capacity. Poly-L-lactic acid (PLLA) and polyglycolic acid (PGA) have emerged as promising biodegradable scaffold materials. The addition of inorganic materials such as calcium carbonate (CC) has also been shown to be advantageous. This study investigates the effect on bone regeneration of PLLA-PGA-CC scaffolds in critical-sized bone defects over a two-year observation period using sheep as an animal model. Methods: Critical-sized mandible angle defects were created in twelve female merino sheep. Mandibular defects were reconstructed with PLLA-PGA-CC scaffolds in four sheep, while the remaining eight served as negative control (defects left empty). The scaffolds were manufactured using computer-aided design and manufacturing, incorporating an interconnected porous structure and fixated with polyether ether ketone cages. Bone regeneration was evaluated using computed tomography (CT) imaging at 3, 12, and 24 months postoperatively. Bone volume was assessed quantitatively. Additionally, a histological analysis was performed. Results: Surgical procedures were successful and without major complications. CT assessment showed more bone regeneration in the scaffold group (mean volume: 7,472 mm3) than in the control group (4,168 mm3, p = 0.1) at 24 months postoperatively. Resorption of the scaffolds and formation of compact lamellar bone tissue were confirmed by histological analysis. However, the osteoconductive properties of the scaffolds were limited, with only minimal ingrowth of bone tissue into the porous structure. In both groups, fibrous tissue infiltration and the formation of cyst-like cavities in the defect region were observed. Conclusion: PLLA-PGA-CC scaffolds were found to be biocompatible and enhanced bone regeneration compared to the control group. Due to fibrous tissue infiltration and the lack of osteoconductivity, the suitability of the material for critical-sized bone defect reconstruction is limited.

The African spiny mouse (Acomys cahirinus) exhibits a unique capacity among mammals for scarless tissue regeneration, making it a compelling model for investigating the cellular mechanisms underlying regenerative healing. To determine how cellular heterogeneity and specific phenotypes influence fibroblast behavior, we established an immortalized Acomys fibroblast line along with a CRISPR/Cas9-mediated Col3A1 knockout variant and a DNA damage-induced senescent population. Compared with Mus musculus, NIH 3T3 fibroblasts, Acomys cells displayed distinct morphology, similar migration speeds, reduced directional persistence, and greater biophysical heterogeneity. While previous studies have linked regenerative wound healing to the elevated expression of collagen type III (Col3A1), CRISPR-mediated knockout of Col3A1 in Acomys fibroblasts yielded comparable biophysical profiles to wild-type cells in 2D culture. To examine additional contributors to the enhanced wound-like matrix environment, we established a senescence model in which Acomys fibroblasts exhibited elevated resistance to DNA-damaging agents, complete loss of proliferation, and altered single-cell morphology. In 3D collagen gel contraction assays, Col3A1 knockout attenuated matrix remodeling capacity, whereas the introduction of a small fraction of senescent cells enhanced gel contraction and remodeling dynamics, suggesting that senescent fibroblasts can modulate collective matrix behaviors. Together, these findings demonstrate that both Col3A1 expression and senescence-associated cell states contribute to fibroblast-driven matrix remodeling, highlighting Acomys fibroblasts as a valuable model for investigating how cellular heterogeneity and senescence-associated cell phenotypes could influence regenerative wound healing.

Angiogenesis drives the formation and remodeling of capillary networks throughout tissue repair, regulating the vascular environment that supports healing and tissue remodeling. Experimental characterization of these processes is commonly performed using CD31-stained histological tissue sections to quantify capillary surface density and morphology throughout healing. However, these measurements provide only two-dimensional characterization of an underlying three-dimensional (3D) vascular network, limiting direct estimation of volumetric capillary density and vascular architecture. To address this limitation, an experimentally informed framework was developed to generate representative 3D capillary networks, enabling estimation of volumetric capillary density from histologically quantified vascular measurements. CD31-stained histological sections obtained from a longitudinal porcine lumpectomy study were analyzed to quantify the percentage of CD31-positive area (%CD31+) and capillary morphology within healthy tissue and healing surgical cavities. Histologically quantified morphology distributions and literature-informed vascular branching characteristics were incorporated into a capillary network generation framework to construct representative 3D vascular networks. Capillary branches were iteratively generated within representative tissue volumes until virtual histological sections reproduced experimental %CD31+ measurements, enabling estimation of volumetric capillary density. Generated capillary networks demonstrated good agreement with experimentally characterized 3D vascular architecture, while simulated histological sections accurately reproduced experimentally quantified capillary counts and vascularization measurements. Application of the framework to the porcine lumpectomy dataset captured temporal changes in vascular remodeling throughout healing, revealing progressive increases in volumetric capillary density and vascular maturation. Collectively, this framework provides an experimentally informed methodology for relating histological vascular measurements to volumetric capillary density estimates, supporting future computational studies of angiogenesis and tissue repair.

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.

Objective: To evaluate the effect of residual hair (RH) biomaterial particulates, biphasic electrical stimulation (ES), and their combination (RHES) on the kinetics and quality of skin wound healing. Method: Eighteen adult albino mice received bilateral, splinted 10-mm full-thickness dorsal excisional wounds and were randomly assigned to one of three animal groups producing four wound-level treatment conditions: untreated control (-) (n = 12), RH (n = 12), ES (n = 6), and combined RHES (n = 6 wounds). Daily wound images were segmented using an AI-assisted workflow: a U-Net (ResNet34 encoder, ImageNet-pretrained, trained on a parallel single-expert tracing study with held-out validation Dice = 0.906) generated initial boundary predictions, each reviewed and corrected as needed. Wound size measures (perimeter, area, equivalent diameter [D_eq], circularity, aspect ratio) were normalized to the day-0 value of each wound and analyzed by linear mixed-effects regression with mouse identity as a random intercept and mouse body weight as a covariate. On day 7, wounds were excised, fixed, processed for histology, and analyzed by Masson's trichrome (collagen content in granulation tissue) and GAP-43 immunohistochemistry (a marker of regenerative cellular activity). Results: All three treatments significantly accelerated wound closure compared to (-) (Day x Treatment interaction {chi}2(3) = 36.4, ***p < 0.0001). The closure-rate advantages on the log-D_eq scale were ES -0.047/day (***p < 0.0001), RHES -0.029/day (***p = 0.0005), and RH -0.022/day (**p = 0.0015). By day 7, mean D_eq had decreased to 0.58 of the day-0 value in ES, 0.69 in RHES, 0.73 in RH, and 0.79 in (-). Tissue analyses revealed treatment-specific differences in healing quality: RH and RHES wounds contained 6.1x and 8.5x more collagen in granulation tissue than (-) (both **p = 0.002 vs (-); both **p = 0.009 vs ES), and showed approximately 16x and 27x greater mean GAP-43 expression than (-), respectively; the RHES increase remained significant after Bonferroni correction (adjusted *p = 0.042), whereas the RH increase did not (adjusted p = 0.058). ES alone did not significantly increase either collagen content or GAP-43 expression. Wound shape was more circular and more stable across days in RH-containing groups. Mouse body weight did not predict closure, whereas image-derived dryness, eschar coverage, and wound contraction were significant negative predictors of measured wound size. Conclusion: ES, RH, and RHES each significantly improve wound closure kinetics. The improvement appears mechanistically distinct: ES principally accelerates closure rate, while RH principally enhances tissue-level regenerative markers (collagen deposition and GAP-43 expression). RHES combines both advantages.

Glioblastoma (GBM) is a highly aggressive brain tumor with a five-year survival rate of less than 5%. The current standard of care established 20 years ago includes maximal surgical resection and administration of alkylating agent temozolomide (TMZ). GBM is highly invasive, and GBM cells that evade surgical resection can become resistant to TMZ and develop new aggressive secondary tumors. Post-relapse there are few treatment options available to patients. Tissue engineering approaches suggest the opportunity to develop in vitro models of the GBM tumor microenvironment that may accelerate the discovery of novel therapies for GBM. Here, we report the adaptation of hydrogel microdroplets (microgels) to encapsulate GBM cells in a tailorable 3D matrix to assess patterns of growth and to screen TMZ drug response using patient-derived xenograft (PDX) specimens. We exploit a unique aspect of the microgel system to account for the cellular heterogeneity within the tumor microenvironment (TME). We combine cell-laden microgels generated from TMZ-resistant and TMZ responsive variants of the same PDX specimens to create heterogeneous populations with varying levels of drug sensitivity. We demonstrate a range of drug resistance phenotypes as a function of the ratio of TMZ-responsive to resistance cells and identify the population required for TMZ-resistance to overtake take the response. We then investigate the influence of tumor mimetic shifts in hyaluronic acid bioavailability and hypoxia on patterns of TMZ resistance. We show exposure to matrix-bound hyaluronan increases TMZ resistance and the glioma stem cell population in both cell variants. Lastly, we report an increase in TMZ sensitivity but divergent changes in the GSC subfraction for TMZ resistant vs responsive GBM in the presence of hypoxia. Together, we demonstrate the versatility of cell-laden microgel approach to replicate heterogenous tumor populations, model shifts in the tumor microenvironment, and rapidly screen therapeutic response.

Constitutive models of myocardial mechanics form a cornerstone of personalized cardiac simulations and cardiac digital twins. Researchers traditionally prescribe these models a priori and calibrate them from ex vivo tissue experiments, even though tissue excision alters loading conditions, removes residual stresses, and eliminates important physiological interactions. Multimodal cardiac MRI now provides subject-specific ventricular geometry, deformation, and myocardial microstructure, yet current inverse approaches still rely on predefined constitutive laws. Here we present the first framework to discover constitutive models of passive myocardial mechanics directly from in vivo cardiac imaging data by embedding a constitutive artificial neural network within a nonlinear finite element model of ventricular filling. Using multimodal cardiac MRI that combines ventricular geometry, deformation, and microstructure from a representative healthy individual, the framework identifies sparse, mechanically admissible strain-energy functions without prescribing their form a priori. The best-performing model contains only two fiber- and two sheet-invariant terms, achieves a mean displacement error of 1.62 mm, and reduces the error of the widely used Guccione and Holzapfel models by 34.14% and 26.01%. The discovered models indicate that fiber- and sheet-related anisotropic mechanisms dominate the passive mechanical response during physiological ventricular filling. More broadly, this work establishes a non-invasive strategy for subject-specific constitutive discovery from cardiac imaging data and lays the foundation for personalized cardiac simulations and cardiac digital twins.

The osteocyte network in bone is believed to play an important role for how bone tissues sense and respond to mechanical stimulation. Yet, bone adaptation to mechanical loads is often conceptualised as a simple response to mechanical stimuli, such as Wolffs law, which is based on mechanical variables only and takes no account of the cellular basis of mechanosensation. Wolffs law presumes the existence of a reference mechanical stimulus, the mechanical setpoint, above which bone is consolidated, and under which bone is removed. In this paper, we develop a theory of bone tissue sensing and adaptation based on osteocytes to provide new understanding of the role played by osteocyte signals in mechanical adaptation. In this theory, the mechanical setpoint of Frosts mechanostat is explicitly embodied as osteocyte properties involved in mechanotransduction. The mechanical setpoint is allowed to adapt due to the replacement of osteocytes during remodelling, making the setpoint space and time dependent. We propose a mathematical model to implement this new theory of bone adapation and present numerical simulations of this model to explore how mechanobiological response curves (effective Wolffs laws) are modulated by setpoint adaptation during remodelling. By accounting for varying osteocyte populations within bone tissue, we explore bone adaptation under osteocyte disruptions, which is particularly relevant to age-related bone loss. Our model suggests that biological disruptions of remodelling balance cannot always be compensated by mechanical feedback, and that setpoint adaptation during remodelling may have significant observable consequences, such as hysteresis in bone response signatures that resemble lazy zones.

Epithelial sheets must maintain robust barrier function while enduring severe mechanical deformations across various physiological environments. While baseline actomyosin contractility is understood to stabilize intercellular junctions and hence cell-cell contact integrity, how cell-generated active forces interact with external physical strain to dictate contact integrity remains poorly understood. In this study, we investigated the biophysical trade-offs between actomyosin contractility and barrier resilience when Madin-Darby Canine Kidney (MDCK) cell islands are subject to large stretch. In contrast to a high concentration (50 M) of the non-muscle myosin II inhibitor blebbistatin that disrupted cell-cell contacts, we first identified a lower concentration (10 M) that maintained cell-cell contact integrity in the absence of any stretch. Such moderate inhibition of non-muscle myosin II reduced, but preserved some level of actin bundle organization. Remarkably, when challenged with a pathological 38% linear stretch using a custom-built biaxial stretching device, 10 M blebbistatin treated epithelial islands exhibited significantly fewer cell-cell contact ruptures than untreated controls, demonstrating a potent protective effect against mechanical strain. Traction force microscopy revealed diminished cell-generated strain energy by over 60% indicating a partial but significant reduction in contractility upon 10 M blebbistatin treatment. Nanoindentation measurements revealed that moderate contractility inhibition decreased the cellular Young's modulus by more than 40%. Consequently, moderate contractility inhibition safeguards epithelial junctions through a dual mechanical effect: it simultaneously reduces baseline active tensile stresses due to cell contractility and lowers the passive elastic forces generated within the softened cell island during external stretch. Our findings indicate that this systemic reduction in forces dominates over any loss of biochemical adhesion strength at cell-cell contacts. We propose that shifting the epithelium from a rigid, highly stressed continuum to a more compliant, relaxed state by moderate contractility inhibition can serve as a general biophysical mechanism to preserve barrier integrity under severe mechanical challenge.

Accurate evaluation of extracellular matrix (ECM) mineralization at the nano-scale is essential for establishing relevant in vitro bone models. This is particularly important with the development and increased application of three-dimensional (3D) cell models for biological research. Transmission electron microscopy (TEM) allows to perform ultra-structural analysis of cells and ECM organization, but its application in in vitro bone models remains limited, due to the potential alteration or loss of the mineral phase during sample preparation. In this study, we compared two TEM sample preparation methods - the conventional chemical fixation and the anhydrous methods - to evaluate their ability to preserve the mineralized ECM in MC3T3-E1 cells cultured as monolayers and as alginate-encapsulated bone spheroids. Chemical fixation preserved cellular ultra-structure and collagen organization, allowing for detailed assessment of cells and ECM organization. Although mineral deposits were detected and their needle-like morphology assessed, characterization of more immature deposits was partially limited by the effects of uranyl acetate and the overall sample preparation process, which could lead to alteration or loss of less stable mineral phases. The anhydrous preparation method resulted in limited preservation of cellular and ECM morphology and did not allow reliable identification of mineral deposits. When applied to spheroids, the chemical fixation method preserved the 3D architecture, collagen-rich ECM and inner mineral deposits, confirming spheroids as a relevant model for bone studies. Overall, these results highlight the need for optimized sample preparation strategies that preserve both ultra-structure and mineral components for accurate nano-scale characterization of bone mineralization.

Adult skin normally resolves injury through rapid closure and fibrotic matrix deposition, often at the cost of permanent appendage loss. We tested whether spatially controlled microtrauma could instead serve as a regenerative entry point when paired with temporally coordinated molecular cues. We engineered a hierarchical extracellular-matrix-based microneedle patch that combines rapid local availability of verteporfin, an inhibitor of YAP-associated mechanotransduction, with sustained retinoic-acid delivery to support follicle-regenerative signalling. The microneedle interface was evaluated in full-thickness rabbit ear wounds, which are prone to hypertrophic scarring, and in Bama miniature-pig wounds, whose skin architecture more closely resembles human skin. Across both models, staged dual-cue treatment accelerated wound closure, reduced collagen-dense scar formation and promoted the appearance of hair-bearing tissue and histologically identifiable follicular structures. These findings support a trauma-guided regeneration framework in which controlled microinjury is used not only for delivery but also to open a transient repair niche that can be molecularly redirected toward appendage-bearing skin restoration. ImportanceMicroneedles are generally treated as minimally invasive delivery devices. Here, the microinjury itself is incorporated into the therapeutic design. The study provides cross-species proof of concept that a patterned injury interface, combined with staged anti-fibrotic and pro-regenerative signalling, can shift wound repair away from fibrotic closure and toward hair-follicle-containing skin. This concise preprint reports the central concept and the rabbit and porcine evidence supporting it; expanded mechanistic and source datasets will be reported separately.

Aquatic animals deploy adhesives, in numerous essential functions, and reversibility is a key adaptation. The molecular mechanisms of reversible wet adhesion remain poorly understood. Using a model organism, the freshwater cnidarian Hydra vulgaris, we dissect the mechanism of molecular assembly in a secreted adhesive and uncover a glycan and protein-based architecture organized by a lectin-like protein, Hydra vulgaris adhesive protein 1 (HvAb1). We identify HvAb1 as a nonredundant organizer of the adhesive matrix, being basal-disc specific and secreted. Knockdown of HvAb1 severely impaired attachment and disrupted footprint architecture in a mosaic pattern, with only HvAb1-positive regions of the adhesive footprint retaining their normal structure. The adhesive is wheat germ agglutinin (WGA)-reactive and contains a fibrillar chitin-based sub-network, synthesized by a basal-disc-specific chitin synthase. Applying exogeneous chitinase abolished both WGA staining and Hydra attachment, indicating that WGA-positive components perform essential roles in adhesion. Our results therefore describe a glycan-dominated matrix, organized via a lectin-like protein (HvAb1), which is reinforced by chitin and enables reversible adhesion underwater. This establishes Hydra as a tractable model to better understand the principles of reversible adhesion underwater and, potentially, inform future bioinspired, sustainable adhesives.