The therapeutic potential of bone marrow mesenchymal stem cells (BMSCs) in bone tissue engineering (BTE) is compromised by functional decline during conventional two-dimensional (2D) expansion. We hypothesized that chitosan film-based three-dimensional (3D) culture rejuvenates BMSC potency, synergistically promoting angiogenesis and immunomodulation for vascularized bone regeneration. Mouse BMSCs were cultured into spheroids on chitosan films. Their stemness, proliferation, migration, senescence, osteogenic, and proangiogenic potential were compared with 2D cultures. Paracrine effects were evaluated by treating human umbilical vein endothelial cells (HUVECs) and RAW264.7 macrophages with BMSC-conditioned medium (CM). In vivo, gelatin methacryloyl (GelMA) hydrogel-encapsulated 3D-BMSC spheroids were implanted into mouse critical-size cranial defects. Compared to 2D counterparts, 3D-BMSCs exhibited increased stemness, proliferation, migration, delayed senescence, osteogenic differentiation, and enhanced proangiogenic potential. We observed that conditioned medium from 3D-BMSCs (3D-CM) was associated with in vitro angiogenesis and orchestrated a proreparative microenvironment by promoting M2 macrophage polarization and suppressing M1 inflammation. In vivo, GelMA + 3D-BMSC spheroids achieved greater bone regeneration, which was accompanied by a proreparative immune microenvironment and enhanced CD31/EMCN-positive H-type-like vessel formation. This chitosan film-based 3D culture system effectively augments BMSC therapeutic potency, simultaneously enhancing intrinsic cell properties and orchestrating a proregenerative microenvironment, thereby offering a promising experimental platform for critical-sized bone defect repair.

Cellular and nuclear morphology, together with directional alignment, are fundamental determinants of cell function and play critical roles in the formation, organization, and maintenance of functional fibrous connective tissues. In tissue-engineered systems, these characteristics emerge from the combined influence of scaffold microarchitecture and mechanical loading, which regulate cellular mechanosensing, cytoskeletal organization, and extracellular matrix remodeling, thereby directly affecting tissue regeneration outcomes. In this study, the coupled effects of scaffold fiber architecture and uniaxial stretching on scaffold organization and cell morphology were investigated using bilayer electrospun scaffolds composed of an aligned-fiber layer (AFL) that gradually transformed into a random-fiber layer (RFL). Scaffold morphology and strain-induced fiber alignment were characterized by scanning electron microscopy and two-dimensional fast Fourier transform analysis at elongation levels of 0%, 10%, and 20%. Adult human dermal fibroblasts were cultured on both AFL and RFL scaffold surfaces, and cellular and nuclear morphologies were quantified using shape descriptors under both unstretched conditions and sustained uniaxial stretching of 10%. Uniaxial stretching promoted alignment of initially random fibers while preserving the orientation of pre-aligned fiber architectures. At the cellular level, nuclear morphology exhibited little dependence on scaffold architecture under unstretched conditions but showed pronounced sensitivity to applied strain. In contrast, cell morphology was governed primarily by fiber alignment and displayed only limited sensitivity to sustained mechanical loading. These findings indicate that scaffold architecture and mechanical stretching regulate structural organization (from scaffold microstructure to cellular and nuclear morphology) through distinct yet complementary mechanisms. The ability to independently and complementarily control structural and mechanical cues offers an effective design strategy for bilayer or graded scaffolds intended for the regeneration of complex tissue interfaces, where both isotropic and anisotropic cellular organization are required.

Peri-implantitis is characterized by bacterial infection and progressive destruction of surrounding tissues, with limited long-term therapeutic strategies. Although macrophages could have therapeutic effects on peri-implantitis, macrophage-based cell therapies have yet to be widely applied in clinical settings. In this study, we developed a macrophage-based approach using hyaluronic acid (HA)-mediated priming and evaluated its in vitro functions and therapeutic efficacy in a mouse model of peri-implantitis. Mouse spleen-derived macrophages were cultured with HA (1.5, 1.8, or 2.0 MDa), and phenotypes were analyzed by Reverse transcription quantitative polymerase chain reaction (RT-qPCR) (inducible nitric oxide synthase [iNOS], tumor necrosis factor-α [TNFα], vascular endothelial growth factor [VEGF], Dectin-1) and flow cytometry (F4/80, VEGF, Dectin-1). Phagocytic and migratory capacities were evaluated, and endothelial cell (HUEhT-2) migration was examined. In vivo, HA-treated macrophages were locally injected into a ligature-induced peri-implantitis model and assessed histologically. HA treatment of macrophages suppressed M1-related (iNOS, TNF-α) and M2a, b, c-related (Dectin-1) gene expression while enhancing the M2d-related gene (VEGF). Flow cytometry revealed an increased proportion of M2d-like cells (Dectin-1[-]/VEGF[+]). HA priming enhanced macrophage phagocytic and migratory capacities and promoted HUEhT-2 migration. In vivo, the HA-treated macrophage group exhibited reduced fibrotic tissue formation and enhanced alveolar bone regeneration, particularly in the MΦ18 group. HA-mediated priming modulates macrophage phenotype and function, supporting its therapeutic potential.Impact StatementThe treatment for peri-implantitis includes infection control and tissue regeneration. Although macrophages exerting anti-infectious and regenerative activities could mitigate peri-implantitis, macrophage-based cell therapies have not yet been widely applied in clinical settings. In this study, we demonstrated the therapeutic efficacy of hyaluronic acid-primed macrophages in a mouse model of peri-implantitis. Our findings not only provide insights into the establishment of new therapies for peri-implantitis but also potentially enhance clinical applications of macrophage-based cell therapies for a wide range of diseases.

Trophoblast invasion into the decidualized endometrium is an important aspect of blastocyst implantation necessary for a successful pregnancy. There are many processes involving the early implantation and invasion of trophoblast cells into the maternal decidua that are not well described. Here, we describe the development of a microfluidic model of the trophoblast-endometrial interface that can be used to quantify metrics of trophoblast cell invasion, endothelial cell motility, and the influence of decidualization hormones on these processes.

Osteoarthritis (OA) is a progressive, degenerative joint disorder characterized by irreversible loss of articular cartilage, in which NLRP3 inflammasome-mediated chondrocyte pyroptosis and dysregulated Wnt/β-catenin signaling are recognized as central pathological events. This study investigated the potential chondroprotective effects of teriflunomide (TFM) in OA, revealing a repositioning molecular mechanism whereby TFM inhibits NLRP3-dependent pyroptosis through the reactivation of Wnt/β-catenin signaling. In vitro, using immortalized human chondrocyte (HC) cells, TFM markedly inhibited interleukin (IL)-1β-induced NLRP3 inflammasome activation and the subsequent pyroptosis relative to that of vehicle-treated control cells. Concurrently, TFM restored Wnt/β-catenin signaling, as indicated by the recovered expression of β-catenin, c-Myc, and cyclin D1. Furthermore, TFM suppressed IL-1β-induced reactive oxygen species generation and speck formation by apoptosis-associated speck-like protein containing a CARD, thus promoting the functional recovery of chondrocytes. These in vitro results were further corroborated in a rat model of OA (n = 6 per group). TFM administration (30 mg/kg, every other day for 4 weeks) improved joint morphology, alleviated synovitis, and restored cartilage thickness and proteoglycan content relative to those observed in vehicle-treated animals. Collectively, these findings suggest that TFM exerts chondroprotective effects in preclinical models by reactivating Wnt/β-catenin signaling and inhibiting NLRP3-dependent pyroptosis. TFM warrants further investigation as a potential candidate agent for the treatment of patients with OA.

The placental barrier plays a critical role in protecting the fetus from xenobiotics and regulating the maternal-fetal exchange. However, conventional trophoblast cell lines and animal models often fail to accurately recapitulate the key structural and functional features of the human placental barrier. In this study, a novel organ-on-a-chip (OOC) model was developed that integrated human JEG-3 trophoblast organoids with human umbilical vein endothelial cells under combined fluid shear stress and mechanical strain. This configuration supports the self-assembly of a three-dimensional, functional placental barrier. The engineered tissue exhibited essential physiological characteristics, including syncytiotrophoblast marker expression, human chorionic gonadotropin secretion, and enhanced glucose transport activity. This model was used to assess the transbarrier transport of several flavonoid compounds. The results indicated that permeation occurred primarily via passive diffusion, with permeability differences governed mainly by physicochemical properties, such as lipophilicity, rather than molecular size. This integrated OOC platform establishes a biomimetic human placental model that provides a reliable tool for evaluating placental permeability and the potential toxicity risks of complex compounds.

Tissue repair and regeneration represent multifaceted processes involving a coordinated interplay of diverse cell types, growth factors, and cytokines. Following tissue injury, innate immune cells serve as the primary cellular responders that infiltrate the damaged microenvironment, clear cellular debris, and undergo phenotype transitions essential for establishing a proregenerative environment. To facilitate functional recovery, three-dimensional scaffolds are conventionally used to give physical signals and structural support. However, these constructs frequently encounter barriers in host-scaffold integration, potential material cytotoxicity, and the induction of adverse chronic foreign body responses (e.g., chronic inflammation), which continue to hinder their successful clinical translation. In this regard, natural polysaccharides, including hyaluronic acid, alginate, and mannans, represent promising biomaterials for multifunctional scaffold engineering due to their extracellular matrix (ECM)-mimetic properties, high biocompatibility, rapid degradation, and tunable physicochemical characteristics. Moreover, the unique sugar moieties inherent to these polysaccharides can function as pathogen-associated molecular patterns and directly interact with pattern recognition receptors expressed on host immune cells. These receptor-ligand interactions trigger intracellular signaling cascades, driving the secretion of downstream cytokines and chemokines, guiding immune cells toward proregenerative phenotypes, and remodeling the ECM to achieve functional tissue repair. In this review, the dual functionality of natural polysaccharides in tissue engineering is described, emphasizing their role as physical supports and active immunomodulatory platforms in regenerative medicine. We critically evaluate the immuno-active motifs within natural polysaccharides, reveal their receptor-mediated interactions with immune cell subsets, and highlight recently reported polysaccharide constructs across distinct tissue targets. We also outline engineering design principles to modulate the chemical, structural, and biophysical properties of immune-active scaffolds. Furthermore, we explore the clinical progress of these scaffolds, addressing current milestones alongside ongoing regulatory and manufacturing challenges. This review provides key design criteria to guide the rational development of next-generation, immuno-instructive polysaccharide constructs for immune-mediated tissue regeneration.

Osteoporosis-associated bone fractures are a leading cause of disability in the elderly population. Developing effective therapeutic strategies to enhance bone repair under osteoporotic conditions remains a major clinical challenge. Increasing evidence indicates that aberrant lineage commitment of mesenchymal stem/stromal cells (MSCs) resident in bone marrow contributes to osteoporosis-related bone loss. However, incomplete understanding of the regulatory mechanisms governing MSC differentiation has limited the development of efficient therapeutic approaches. In this study, we identified micro-RNA-423 (miR-423) as a negative regulator of osteogenic differentiation, and demonstrated that inhibition of miR-423 significantly enhanced osteoblast differentiation of MSCs. To enable in vivo delivery of the miR-423 inhibitor for bone repair, MSC-derived exosomes (MSC-Exo) were used as a delivery vehicle, generating the Exo-miR-423 inhibitor construct. These exosomes were subsequently incorporated into an apatite-coated poly(lactic-co-glycolic acid) scaffold to form an Exo-miR-423 inhibitor/scaffold complex. Implantation of this complex significantly promoted bone healing in a calvarial defect model in ovariectomized mice. Collectively, these findings demonstrate a promising miRNA-modulated, exosome-based tissue engineering strategy for enhancing bone defect and fracture repair under osteoporotic conditions, and highlight its potential for further optimization and translational application.

Although embryonic chondrogenesis follows conserved developmental stages across mammalian species, the rate of this process varies significantly. We hypothesized that these varying developmental rates could be normalized and modeled by aligning key developmental milestones of articular cartilage: mesenchymal condensation, chondrogenic differentiation, joint interzone formation, cavitation, and primary ossification center formation. A mathematical model was developed in MATLAB by fitting the developmental curves of these key stages in mice, rats, pigs, horses, and humans. Among various mathematical models, a sigmoid curve model demonstrated the best fit, with the highest R[2] (0.98 ± 0.02) and lowest residual norm (0.36 ± 0.21) after normalizing for gestation period and femoral condylar width to account for species-specific differences. The model's high predictive accuracy (R[2] = 0.98) was further validated using data from rabbits, canines, and sheep. By differentiating the developmental curve equation, we determined the developmental rate for each species. Our results indicate that embryonic chondrogenesis occurs within a relatively narrow window of gestation (11-32%), with rodents exhibiting faster rates than larger animals. This study presents the first mathematical model to quantitatively align embryonic chondrogenesis across mammals. This model has the potential to fill gaps in developmental timelines through rational calculation and by improving the precision of developmental timing, factors that have often been overlooked in the field. As a pioneering effort, this work opens new avenues for cross-species alignment and enhances the accuracy of data related to embryonic chondrogenesis.Impact Statement As the first mathematical model to align chondrogenesis across different mammals, this work has the potential to enhance the consistency and accuracy of data from various species by correcting inaccuracies and supplementing missing information.

Normothermic machine perfusion (NMP) has emerged as a new technique for organ storage before liver transplantation, making viability testing of the organ feasible and possibly allowing for reconditioning of marginal liver grafts. Erythrocytes form a substantial component in the composition of NMP perfusates. The optimal hematocrit (HCT) for preserving organ function during NMP and the effects of the HCT on metabolic graft function and parameters commonly used to determine organ viability are still unclear. We retrospectively analyzed our rat NMP database of 123 perfusions and identified a possible link between HCT and liver damage parameters in 34 cases following a dual perfusion protocol. To further investigate this relationship, we perfused a total of 16 rat livers for 6 h each in our rat liver NMP setup with four different HCTs at 10%, 16%, 22%, and 28%. We performed blood gas analysis and measured common viability and damage parameters such as bile production, lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). In addition, histological analysis, as well as enzyme-linked immunosorbent assay and colorimetric assay analysis for reactive oxygen species, glycogen content, and hypoxia-inducible factor (HIF), was performed. The perfusate HCT significantly impacted ALT and LDH levels during NMP of rat liver grafts, with lower HCT values corresponding to decreased damage parameters. We investigated differentiating oxygenation levels and identified a possible cause for our observations in HIF-1α stabilization in lower HCT groups. Furthermore, we demonstrate that HCT affects key viability parameters such as lactate, pH levels, and bile production. Our findings illustrate the importance of adequate HCT levels during NMP and propose mechanisms influencing the perfusion outcome.Impact StatementNormothermic machine perfusion (NMP) is becoming an increasingly important tool in liver transplantation. We here show in an exploratory setting the importance of an adequate hematocrit for the outcome of liver graft perfusion and its importance in defining hepatic viability parameters during NMP.

Urethral strictures can cause significant discomfort and progressive urinary tract damage if left untreated. Current reconstructive options, including urethral resection and buccal mucosa grafting, are associated with several limitations such as donor-site morbidity, limited tissue availability, and variable long-term outcomes. To address these challenges, we developed a novel multilayered 3D-bioprinted urethral construct designed to closely mimic the native urethral architecture. Using an Integrated Tissue and Organ Printing System (ITOP) equipped with a rotating mandrel, tubular urethral constructs were fabricated with distinct layers consisting of urothelial cells (UC), basement membrane (BM), smooth muscle cells (SMC), and supportive polycaprolactone (PCL). Autologous UC and SMC isolated from urinary bladder tissue were incorporated into a fibrinogen-based hydrogel bioink. Following in vitro maturation, the constructs were evaluated using viability assays, immunohistochemistry, and biomechanical testing. Live/Dead staining demonstrated an average cell viability of 75% for both UC and SMC populations. Immunostaining confirmed appropriate localization of the different cell types within their respective layers. Tensile testing showed that constructs matured for 14 days developed stable and elastic tissue-like mechanical properties. To further improve construct handling and structural integrity, horizontal reinforcement bands were incorporated into the PCL layer. Using this approach, 4 cm-long and 0.5 cm-diameter urethral constructs with native-like multilayered organization were successfully fabricated. This novel 3D-bioprinting strategy demonstrates strong potential for generating customizable and biologically relevant urethral grafts for future reconstructive applications. Ongoing in vitro optimization and planned in vivo evaluation in a porcine model aim to further validate the structural and functional performance of the construct and support future clinical translation for urethral and other tubular tissue reconstruction.

Severe burn injuries pose significant clinical challenges, with high risks of infection, excessive inflammation, and impaired wound healing. Mesenchymal stem cells (MSCs) have shown regenerative and antimicrobial potential; however, their therapeutic efficacy is constrained by poor survival and engraftment. Here, we demonstrate that priming MSCs with insulin-secreting cells (ISCs) and encapsulating them in hydrogels (HEMI) enhances their regenerative function, leading to accelerated healing of full-thickness burns in a porcine model. By 5 weeks, 80% of HEMI-treated wounds achieved complete closure, compared with 50% of MSC-only wounds and 0% of standard-of-care control wounds. All groups exhibited partial wound closure over time, but no control wounds reached complete closure within the study period. Histological analysis revealed complete epidermal and dermal regeneration with minimal fibrosis in HEMI-treated wounds. Single-cell RNA sequencing and differentially abundant sequencing analysis identified distinct MSC subpopulations whose relative abundance and transcriptional profiles differed between insulin-primed and control MSCs, with insulin priming promoting pathways involved in extracellular matrix stabilization, immune modulation, and oxidative stress resistance. Our findings suggest that insulin priming enhances MSC-mediated tissue repair via paracrine mechanisms, providing a clinically translatable strategy for improving burn treatment and regenerative medicine applications.

To develop bone grafts with superior osteoconductivity, we have fabricated unidirectionally connected porous carbonate apatite (CAp) via gelatin gelation combined with freeze-drying. A composite of calcium carbonate (CaCO3) and gelatin was first gelled at low temperature and then subjected to directional freeze-drying to create well-aligned macroporous channels. The samples were then sintered to remove the organic components and subsequently phosphatized to convert the composition into CAp. The resulting material exhibited a porosity exceeding 80% with fully interconnected unidirectional pores favorable for cell migration and vascular infiltration. Bone regeneration was evaluated by implanting the material into rabbit femoral bone defects; a comparison was also made with nonoriented porous controls. Histological and radiographical analyses demonstrated that the unidirectional porous structure significantly enhanced directional ingrowth of new bone tissue and accelerated early-stage bone formation relative to the control. This study demonstrates that unidirectional porous CAp fabricated via a gelatin-based freeze-casting method is promising for bone regeneration, particularly in clinical applications requiring guided bone formation.

Chronic tympanic membrane (TM) perforation remains difficult to resolve without surgery because of poor intrinsic healing capacity and limitations of passive, nonsurgical materials. Although patch-based approaches are common, most merely provide passive coverage and lack intrinsic therapeutic capability to drive tissue regeneration. We developed and clinically evaluated an extracellular matrix (ECM)-mimetic, drug-free nanopattern guidance (NG) patch for TM repair, marking a paradigm shift from passive coverage to active, biophysically driven regeneration. The NG patch is a biocompatible, implantable scaffold with aligned nanotopography that recapitulates native ECM architecture, coupled with a hydrocolloid adhesive layer to ensure secure placement. In a prospective study of 18 patients, the NG patch achieved a 61% overall healing rate with 50% complete closure and no serious adverse events. In rat models, a single application induced complete healing within 2 weeks. Mechanistic in vitro assays demonstrated that the nanopatterned surface enhances fibroblast adhesion, alignment, and directional migration, supporting organized tissue closure. Collectively, the NG patch offers a minimally invasive approach that activates TM repair through physical cues without exogenous drugs. This first clinical evaluation of an ECM-mimetic nanotopographical scaffold introduces a strategy to redefine the standard of care for chronic TM perforation and related defects.