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.
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.
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.
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.
Pelvic organ prolapse (POP) is an urgent clinical challenge, and traditional surgical treatment is limited by high recurrence rate, erosion, mesh exposure, pain, and other complications. Recently, umbilical cord mesenchymal stem cells (UMSCs) have emerged as a promising modality for tissue regeneration and Pelvic floor repair. This study aimed to assess the effectiveness of polypropylene mesh (GynemeshTM PS) loaded with human UMSCs (HUMSCs) primed with IFN-γ for the treatment of POP. In this study, an ideal IFN-γ concentration was chosen to stimulate HUMSCs and create an enhanced version by incorporating the activated HUMSCs onto the mesh. Balloon distension was used to create a rat POP model, and the mesh was implanted into the vaginal wall of rats. The meshes were removed at week four after implantation in order to assess the treatment effect. The improved mesh displays good biocompatibility. In vitro results indicate that IFN-γ stimulates HUMSC proliferation and enhances paracrine effects. In vitro results also demonstrate that it effectively reduces inflammation and promotes angiogenesis, collagen deposition, and cell proliferation, thereby accelerating tissue repair. Overall, this innovative therapeutic approach offers a new avenue for POP.
In tendons, ligaments, and menisci, collagen fibers running the length of the tissue are the primary source of strength and function. Cells assemble these fibers hierarchically from nanometer-wide fibrils into larger fibers and fascicles, increasing in size throughout development and with mechanical loading. These fibers largely do not regenerate after injury or with repair, limiting recovery options. Engineered replacements are a promising treatment option; however, it remains a challenge to produce the hierarchical collagen fibers essential to tissue strength, limiting their applications. To better repair, regenerate, and engineer these tissues, we must better understand how cells regulate hierarchical fiber formation and maintenance. It is well established that mechanical cues are critical for cell-driven hierarchical fiber formation, which cells sense through several mechanisms, such as integrin-mediated adhesions, cell-to-cell connections, mechanosensitive ion channels, primary cilia, and caveolae. These mechanisms of mechanosensation have been well studied at the fibril scale of collagen organization, but as tissues mature, the loading environment becomes more complex, with cells experiencing increasing secondary shear and compressive loads generated by the developing hierarchical structure. Mechanical cues in this environment are likely sensed through several pathways, each likely playing a role in tissue maturation and injury. There remains a clear gap in our understanding of the later stages of hierarchical fiber formation, which are crucial to better understand since large hierarchical fibers dominate the human musculoskeletal system. Here, we review the role of mechanobiology in hierarchical fiber development and maintenance, and highlight what still needs further research to better regenerate fibers in engineered replacements or in vivo after injury. A better understanding of the mechanisms by which cells form hierarchically organized collagen fibers could help to overcome the limitations of current tissue engineering techniques and help to create functional repairs and replacements.
Because articular cartilage (AC) lacks inherent repair capacity, research has focused on translating tissue-engineered cartilage to the clinic. Toward this, rapid and nondestructive methods would be useful for determining in-process and release characteristics during the manufacture of tissue-engineered products. The current work aims to introduce a Raman-based methodology for nondestructive qualitative and quantitative characterization of tissue development using AC. First, Raman shifts associated with critical biochemical components of AC, with particular emphasis on DNA, glycosaminoglycans (long chains of sugar molecules and a key component of cartilage), total collagen, as well as pyridinoline (a marker of collagen crosslinking and maturation), were collated. Next, verification of the molecular spectroscopic biomarkers was conducted by temporally tracking tissue maturation/development of nascent and mature AC, establishing a temporal reference dataset. Finally, validation was performed by correlating the spectroscopic biomarkers with traditional photometric biochemical assays and mass spectrometry. The results presented here include a Maturity Index for quantification of tissue development/maturation. Strong correlations were found between nondestructive spectroscopic-based measurements and destructive (photometric and mass spectrometric) measurements with high linearity for both nondestructive Raman (R2 > 0.96) and destructive biochemical (R2 > 0.97) assays, respectively. Uses of the proposed rapid and nondestructive method include in-line quality assessment (in which the sample is not removed from the process stream) to monitor the manufacturing of tissue-engineered medical products. This study shows that Raman spectroscopy has the capacity of being a powerful tool for nondestructive quality control and assurance in traditional biomanufacturing workflows, and the approach taken here may also be utilized as a template and research tool for studies on the development of other native and engineered tissues.
Volumetric muscle loss (VML) injuries result in an irrecoverable loss of muscle mass and function. VML injury causes loss of both contractile tissue and associated neuromuscular junctions (NMJs). Biosponge (BSG) scaffolds, composed of gelatin, collagen, and laminin-111, have improved recovery following VML. However, improvements in NMJ quantity were not observed. Glial cell line-derived neurotrophic factor (GDNF) is known to promote motor unit survival and stimulate neurite outgrowth. In this work, BSG scaffolds were electrostatically coupled with GDNF via gelatin nanoparticles (GNPs) to support myofiber regeneration and preserve NMJs post-VML in a rodent model. In vitro determination of release kinetics revealed an initial burst release of surface-bound GDNF with almost an equivalent amount of electrostatically bound GDNF retained within the BSG post 1 week of incubation at 37°C in phosphate-buffered saline. To create the VML injury in male Lewis rats (10-12 weeks old), ∼20% of the muscle mass was removed from the tibialis anterior (TA) muscle of both hindlimbs. Relative to BSG + GNP alone, treatment with BSG + GNP + GDNF showed a significant increase (∼25%) in peak isometric torque at 6 weeks postinjury. Qualitative and quantitative histological analysis of NMJs revealed an enhanced overlap between pre and postsynaptic structures in the BSG + GNP + GDNF group. Additionally, the incorporation of GDNF slowed BSG remodeling and degradation. Overall, these results suggest that BSG-mediated GDNF delivery is an effective strategy for mitigating NMJ loss and enhancing muscle recovery following VML.
Bone is a common site of breast cancer metastasis, which dramatically increases fracture risk. Recent in vivo bone metastasis studies show that mechanical loading is osteoprotective; however, little is known about how loading regulates breast cancer cell function in the unique bone mechanical environment, especially in combination with radiotherapy, one of the first-line treatments for advanced breast cancer patients. Here, we characterize the breast cancer cell response to a range of bone-mimicking fluid shears and determine how irradiation further modulates one candidate gene: SERPINE1. We found that irradiation, regardless of dosage, modulates SERPINE1 expression and is sensitive to the timing of administration. Additionally, protein expression of SERPINE1 accompanies a protumorigenic gene expression profile, which is elevated with higher-magnitude fluid shear stresses in a bone-mimicking 3D environment. Thus, we postulate that plasminogen activator inhibitor-1 (PAI-1) (encoded by SERPINE1) is a critical growth factor contributing to osteolytic lesion development in the bone metastatic vicious cycle of breast cancer.
The Coronavirus Disease of 2019 (COVID-19) pandemic caused by SARS-CoV-2 resulted in widespread mortality, economic strain, and healthcare system disruption, highlighting the need for effective strategies to address viral threats. Tissue engineering and biomaterials can contribute significantly in advancing our understanding and treatment of respiratory viral infections by developing physiologically relevant in vitro models, controlled and targeted drug delivery systems, and effective next-generation vaccine platforms. Over the last 5 years, tissue-engineered cell-based models, including organ-on-a-chip systems, have improved our understanding of viral entry, immune response, and therapeutic efficacy in pulmonary, cardiac, neurological, and vascular tissue. Biomaterial-based delivery systems have enhanced the targeting, bioavailability, and sustained delivery of therapeutics such as antivirals, monoclonal antibodies, nucleic-acid-based vaccines, and anti-inflammatory drugs, while also reducing or eliminating system toxicity and our reliance on intravenous administration. Advancements in vaccine platforms using lipid nanoparticles, protein scaffolds, and vesicular or cell-based platforms are in different phases of development to stimulate more robust responses against emerging SARS-CoV-2 variants. Collectively, these advancements highlight the influence of tissue engineering and biomaterials in SARS-CoV-2 research and treatment. This review provides an overview of recent developments in in vitro modeling, drug delivery systems, and vaccine platforms, highlighting their future role in improving clinical outcomes, managing variants, and preparing for potential future pandemics, including addressing challenges in infrastructure such as limited access to high-containment biosafety facilities used to study emerging infectious pathogens.Impact StatementDuring the COVID-19 pandemic, we introduced basic tenets of virology to a tissue engineering audience and proposed different areas in which the field could contribute to developing diagnostics and therapeutics for respiratory viral infections. In this 5-year update, we highlight how tissue engineers and biomaterial scientists contributed tools to dissect virus pathophysiology and deliver therapies such as mRNA vaccines to prevent mortality during the pandemic. With the emergence of new variants and the threat of new respiratory viral pandemics, tissue engineers can continue to play important roles in virology.
Maternal and fetal health during pregnancy is closely tied to contractile modulation of the uterine myometrium, as dysfunctional contractions underlie pregnancy complications such as preterm labor and postpartum hemorrhaging. Structural abnormalities increase the risk for pregnancy complications, yet the complex interplay between biochemical and biomechanical cues in the myometrial microenvironment is not well defined. Significant gaps in our understanding of myometrial physiology and the lack of physiologically relevant research tools to enable systematic investigation have resulted in a largely ineffective and nonspecific therapeutic landscape for myometrial contractile modulation during pregnancy. We address this unmet need by developing a widely accessible approach for in vitro hydrogel platform fabrication to advance the study of agonist-mediated calcium responses in myometrial cells. We directly fabricated polyacrylamide hydrogels in polypropylene multiwell plates and Petri dishes and validated their mechanical properties and use as a mechanically tunable cell culture substrate. Using "physiological" matrix conditions during pregnancy, a fluorescent calcium mobilization assay was implemented in a plate-reader-based workflow to determine the dose sensitivity of myometrial cells to the endogenous agonist oxytocin. Using hydrogels with elastic moduli spanning physiological, pathological, and supraphysiological conditions in the myometrium, we assessed stiffness-mediated effects in myometrial cell behavior, including agonist-mediated calcium responses. We observed the mechanosensitive modulation of calcium response curve amplitude in oxytocin-stimulated myometrial cells. Myometrial cell behavior on mechanically tunable substrates was compared against widely used tissue culture plastic, and the observed changes to myometrial cell morphology and calcium responses highlight the significant influence of supraphysiological substrates. To explore complex pathological microenvironments, myometrial cells were exposed to the proinflammatory trigger lipopolysaccharide (LPS) before agonist stimulation. In our studies, LPS exposure altered calcium responses during oxytocin stimulation in cells cultured on supraphysiological substrates. Altogether, we present an easily adoptable in vitro hydrogel platform with scalable fabrication and versatile application use, including new directions in myometrial mechanobiology.Impact StatementWe investigated mechanosensitive modulation of agonist-mediated calcium responses in myometrial cells during pregnancy using a high-throughput in vitro polyacrylamide hydrogel platform. Polypropylene labware enabled scalable fabrication of an in vitro hydrogel platform without being costly or labor-intensive. Our studies found that oxytocin-stimulated calcium responses in myometrial cells are sensitive to nonphysiological matrix conditions. This work highlights the importance of using physiologically relevant engineered microenvironments and represents new directions for understanding myometrial mechanobiology. Our platform can help accelerate the identification of agonists and antagonists to counteract dysfunctional myometrial contractions and can be used more broadly in other tissues that exhibit mechanosensitive pathologies.
Critical-sized bone defects remain a significant clinical challenge, as their size prevents spontaneous healing and necessitates surgical intervention. Although autografts are considered the clinical gold standard, their use is limited by donor site morbidity and tissue availability, while allografts carry risks of disease transmission and long-term failure. In this study, we developed a composite polymer ink for high-resolution digital light processing (DLP) 3D printing of bone tissue engineering scaffolds intended for load-bearing applications. A combination of poly(propylene fumarate) (PPF), poly(caprolactone fumarate) (PCLF), and hydroxyapatite (HA) was formulated to achieve tunable mechanical properties and controlled scaffold architecture. Scaffolds with varying porosities and material compositions were fabricated and evaluated using compressive mechanical testing and finite element modeling to assess structural integrity and stress distributions. In vitro studies using preosteoblast cells demonstrated consistently high cell viability (>75%) across all scaffold designs, with sustained proliferation over 7 days. Notably, scaffold porosity and material composition influenced proliferative responses, with significant increases observed in select formulations. Collectively, these results demonstrate that DLP-printed PPF/PCLF/HA composite scaffolds provide a mechanically viable and cytocompatible platform with tunable properties, supporting their potential utility in bone tissue engineering applications.Impact StatementCritical-sized bone defects lack effective, widely accessible treatment options due to the limitations of current grafting strategies. This work introduces a digitally light processed (DLP) 3D-printable composite scaffold with tunable mechanical properties and architecture suitable for load-bearing applications. By integrating poly(propylene fumarate), poly(caprolactone fumarate), and hydroxyapatite, the platform enables control over structural and biological performance while maintaining high cytocompatibility. These findings highlight a scalable and customizable approach to bone tissue engineering that may reduce reliance on traditional grafts and improve outcomes in complex bone repair.
Despite advances in tissue engineering, the clinical application of scaffolds for meniscal repair remains limited by implantation challenges and the difficulty of directing stem cells toward a stable meniscal phenotype. Here, we present an injectable hydrogel composed of silk fibroin (SF) and tannic acid (TA), codelivering transforming growth factor-β3 (TGF-β3) and the transcription factor Mohawk (MKX). TA enhanced the β-sheet content of SF, yielding a hydrogel with superior rheological stability and shear-thinning behavior suitable for minimally invasive administration. The ST hydrogel provided sustained release of both bioactive factors over 35 days, with cumulative release below 16%. MKX attenuated TGF-β3-induced hypertrophic differentiation of adipose-derived mesenchymal stem cells, promoting a fibrochondrocyte-like phenotype characterized by elevated expression of COL1A1, COL2A1, and aggrecan. Inflammatory stimulation assays demonstrated downregulation of osteoarthritis-related genes (e.g., MMP13, IL-6, and RUNX2) in the ST-TM group. In a rabbit meniscus tear model, ST hydrogels loaded with TGF-β3 and MKX significantly enhanced extracellular matrix deposition, promoted structural repair of the meniscus, and improved biomechanical properties at 10 weeks postimplantation. These findings support the potential of this dual-factor hydrogel system as a cell-free therapeutic strategy for functional meniscus regeneration, with potential relevance to the modulation of OA-related degeneration.Impact StatementThis study presents a novel injectable silk fibroin/tannic acid hydrogel codelivering transforming growth factor-β3 and Mohawk, which synergistically promotes fibrochondrocyte differentiation and inhibits hypertrophy of stem cells. Demonstrating injectable handling characteristics, controlled release, and in vivo efficacy, this cell-free platform offers a clinically translatable strategy for functional meniscus repair, with potential protective effects against osteoarthritis-related degeneration.
This study aimed to scrutinize the antifibrotic effects of jujuboside A-loaded exosomes (JuA-Exo) on TGF-β1-induced MRC-5 fibroblasts and explore the pathophysiological basis. Exosomes were isolated from human mesenchymal stem cells and loaded with JuA by ultrasonic dispersion. A TGF-β1-induced MRC-5 fibrosis model was established. Cell viability, migration capacity, levels of inflammatory cytokines, extracellular matrix components, and autophagy-related markers were assessed. To determine autophagy dependence, the autophagy inhibitor 3-methyladenine (3-MA) was employed. JuA-Exo significantly attenuated fibroblast activation triggered by TGF-β1, decreased the secretion of IL-6, IL-1β, and hydroxyproline, and downregulated fibrotic gene expression, including α-SMA, fibronectin, COL1A1, and COL3A1. Mechanistically, JuA-Exo was associated with modulation of the TGF-β/Smad signaling pathway and restoration of autophagy-related markers. Importantly, the defensive effects of JuA-Exo were reversed upon autophagy inhibition by 3-MA. JuA-Exo exerts antifibrotic and anti-inflammatory effects, which are associated with modulation of TGF-β/Smad signaling and autophagy-related processes; however, the mechanistic linkage between these pathways and their causal roles requires further validation. These findings highlight JuA-Exo as a potential experimental candidate, although the current evidence is limited to in vitro observations and does not yet support therapeutic application.Impact StatementThis study demonstrates that jujuboside A-loaded exosomes exert antifibrotic and anti-inflammatory effects in vitro, providing mechanistic insight and highlighting a potential nanotherapeutic approach that warrants further validation.
Traditional approaches to tissue-engineered small intestine (TESI) require implanting a construct in the omentum of the abdominal cavity to allow for vascularization of the construct and tissue maturation. A second operation is then needed to implant the construct in continuity with the intestine. The timing of the second operation has historically been chosen based on predetermined biological endpoints. Currently, no well-validated method has been used to noninvasively monitor for vascular maturity which would better inform when the second operation should be performed. We hypothesized that photoacoustic imaging (PAI) could serve as a tool to noninvasively monitor regional and temporal changes in vascular maturity in scaffolds implanted in the omentum. For this pilot study, tubular scaffolds used for TESI were fabricated with electrospinning and implanted in the omentum of Sprague-Dawley rats for 1 and 2 months. Scaffolds were imaged with PAI and tissue oxygenation and hemoglobin concentration were quantified. PAI was then correlated with gross observations of vascularization at each time point. PAI was able to determine regional and temporal changes in tissue oxygenation and hemoglobin concentration. Specifically, the oxygenation and hemoglobin concentration of the top wall of the construct showed better vascular maturity compared with the bottom wall. In addition, vascular maturity seemed to improve in the top wall from 1 to 2 months. The bottom wall was not well covered by the omentum and thus did not vascularize as well as the top wall. The PAI findings were confirmed on gross examination of the scaffolds and upon quantification of the histological analysis of endothelial cell density. Thus, PAI may serve as a critical tool for monitoring vascular maturity in TE, specifically within the abdomen. This will be a critical tool in the preclinical development and clinical translation of TESI.Impact StatementImaging methods to track functional changes in tissue-engineered constructs are need. These critical tools will be essential for tissue-engineered small intestine (TESI) development and clinical translation. In particular, understanding when a TESI construct has achieved vascular maturity is required prior to placing the TESI in connection with native intestine. In this proof-of-concept study, we have shown that photoacoustic imaging can be used to noninvasively track construct vascular maturity and that the imaging correlates with end-point histology. In addition to TESI, this tool is broadly applicable to all tissue-engineered constructs.
