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Bone defects in the maxillofacial region severely impair patient function and esthetics. Free autologous bone grafting remains the gold-standard treatment; however, surgical intervention at donor sites limits clinical applicability. Treatment using artificial materials also presents challenges, including insufficient bone regeneration and poor biocompatibility. Bio three-dimensional (3D) printing, which enables the fabrication of scaffold-free 3D constructs from cellular spheroids has emerged as a promising regenerative approach. This study investigated the osteogenic potential of scaffold-free constructs composed of human dental pulp stem cell (DPSC) spheroids in a rat mandibular defect model. DPSCs isolated from extracted human teeth were used to generate spheroids, which were assembled into 3D constructs using a Bio 3D printer. The spheroids exhibited higher mRNA expression of stem cells and early osteogenic markers than monolayer cultures. The constructs were transplanted into mandibular defects of immunodeficient rats, and bone regeneration was assessed eight weeks post-transplantation. Radiographic and micro-Computed Tomography analyses revealed significantly greater bone volume and mineral density in the 3D construct group. Histological and immunohistochemical examinations confirmed newly formed bone containing osteogenic cells derived from the transplanted DPSCs. These findings indicate that Bio 3D-printed, scaffold-free DPSC constructs promote mandibular bone regeneration and may provide a novel strategy for maxillofacial reconstruction. Full article

New nano-biomaterials for specific dentistry applications have been developed thanks to contributions from materials science. The present work aims to characterize the physicochemical properties of a composite nanomaterial scaffold in block form for maxillofacial bone regeneration applications. The scaffold was composed of block-shaped elements and consisted of a mixture of nano-hydroxyapatite, β-tricalcium phosphate, and type I collagen of bovine origin. Collagen I molecule is biodegradable, biocompatible, easily available, and a natural bone matrix component. The biomaterial was analyzed using a range of methods, including scanning electron microscopy (SEM), transmission electron microscopy (TEM), chemical composition microanalysis, and X-Ray diffractometry (XRD). The wettability was measured. This was carried out by measuring the contact angle of a 0.9% NaCl solution on the surface. Differential scanning calorimetry (DSC) was used to measure the phase transformation temperatures. In the SEM and TEM analyses, it was possible to identify the layers of the materials and, with microanalysis, quantify their chemical composition. The XRD spectra showed the presence of nano-hydroxyapatite and ß-TCP. Wettability testing revealed that the material is highly hydrophilic, and BM-MSC culture analyses demonstrated that the biomaterial can promotes cell adhesion and interaction. The higher wettability is due to the higher density of the porous material observed in the SEM analysis. The results of the DSC testing showed that the sample analyzed undergoes endothermic transitions and transformation between 25 and 150 °C. The first phase transformation during heating occurs at 61.1 °C, which is above body temperature. The findings demonstrated that the composite was devoid of any contamination arising from manufacturing processes. It can be concluded that the n-HA/β-TCP and type 1 collagen are free of manufacturing contaminants. They also have high wettability, which increases the spreading of body fluids on the biomaterial’s surface and its interactions with cells and proteins. This makes them suitable for clinical application. Full article

The design of biomaterial scaffolds for bone tissue engineering requires a balance between bioactivity, porosity, mechanical stability, and osteoinductivity. Kappa- (KC) and iota-carrageenan (IC) have been explored for scaffold fabrication due to their biocompatibility and structural similarity to glycosaminoglycans. However, there are limited reports on how their distinct sulfation degree affects the osteogenic differentiation of cells cultured on them. While laponite has been reported as an osteoinductive nanoclay, its combined effect with different carrageenan types and its concentration-dependent effect on scaffold functionality remain unexplored. Therefore, we developed composite scaffolds comprising poly(vinyl alcohol) (PVA) and gelatin (GEL), reinforced with kappa- or iota-carrageenan (KC, IC) and functionalized with two different concentrations of laponite (LAP), 0.5 and 1% w/v, to monitor composition-structure-function relationships. The scaffolds were fabricated via lyophilization and dual crosslinking, and characterized for their physicochemical, structural, mechanical, and biological properties. The incorporation of both carrageenans into scaffolds, maintained high swelling ratios of 600% after 24 h, and increased porosity without altering their apparent density (0.09–0.11 g/cm³), whereas LAP preserved interconnectivity, densified pore walls, raised their compressive modulus at >220 kPa, and improved stability (>60% mass retained after 40 days). In vitro validation using MC3T3-E1 pre-osteoblastic cells demonstrated robust cytocompatibility, with the LAP-containing scaffolds significantly promoting cell adhesion, proliferation, and osteogenic differentiation, evidenced by elevated alkaline phosphatase activity, calcium production and collagen secretion. Direct comparison between KC and IC scaffolds confirmed that differences in sulfate substitution modulated scaffold stiffness, swelling, and degradation, while variation in LAP concentration affected the biological response, with the 0.5 wt% concentration favoring early cell proliferation, whereas the 1 wt% significantly promoted the osteogenic differentiation. This compositional strategy demonstrates how tuning the interplay between carrageenan and laponite can balance scaffold hydration, mechanical and biological properties, thereby guiding the design of scaffolds for bone repair. Full article

Biomaterials made with extracellular matrix obtained from allogeneic or xenogeneic tissues/organs or cultured cells have excellent biochemical and physical properties in supporting cell growth and tissue regeneration. These decellularized extracellular matrix-based biomaterials have been applied in clinical trials and have bright prospects in tissue engineering and regenerative medicine. Here, we systematically compare organ-derived and cell-derived decellularized extracellular matrix, summarize commonly used decellularization methods, including physical, chemical, and biological/enzymatic treatments, as well as combinations of these treatments, and characterize methods for decellularization, including histological staining, immunohistochemical techniques, biochemical analysis, scanning electron microscopy, and mechanical stress testing. Besides the production of decellularized extracellular matrix, the evolving intellectual property landscape and commercial products are also introduced. A significant focus is placed on summarizing clinical trial outcomes, demonstrating the efficacy of decellularized extracellular matrix scaffolds in diverse applications, including wound healing, cardiovascular repair, nerve regeneration, and breast reconstruction. Finally, we discuss persistent challenges and future directions, underscoring the translational potential of decellularized extracellular-matrix-based strategies for restoring tissue structure and function. Full article

Macrocyclic drugs are promising for targeting undruggable proteins, including those in cancer. Our prior work identified BE-43547A₂ (BE) as a selective inhibitor of pancreatic cancer stem cells in PANC-1 cultures, but its high lipophilicity limits clinical application. To address this, we designed derivatives retaining BE’s backbone while modifying tail groups to improve its properties. A concise total synthesis enabled a versatile late-stage intermediate (compound 17), serving as a platform for efficient diversification of BE analogs via modular click chemistry. This approach introduced a central triazole ring connected by flexible alkyl spacers. Key properties, including lipophilicity, solubility, and Caco-2 permeability, were experimentally determined. These derivatives exhibited reduced lipophilicity and improved solubility but unexpectedly lost cellular activity. Direct target engagement studies using MicroScale Thermophoresis (MST) revealed compound-dependent deactivation mechanisms: certain derivatives retained binding to eEF1A1 with only modestly reduced affinity (e.g., compound 29), while others showed no detectable binding (e.g., compound 31). Microsecond-scale molecular dynamics simulations and free-energy calculations showed that, for derivatives retaining target affinity, tail modifications disrupted the delicate balance of drug–membrane and drug–solvent interactions, resulting in substantially higher transmembrane free-energy penalties (>5 kcal/mol) compared to active compounds (<2 kcal/mol). These insights emphasize the need to simultaneously preserve both target engagement and optimal permeability when modifying side chains in cell-permeable macrocyclic peptides, positioning compound 17 as a robust scaffold for future lead optimization. This work furnishes a blueprint for balancing drug-like properties with therapeutic potency in macrocyclic therapeutics. Full article

Microfluidic cell culture systems and organ-on-a-chip platforms provide powerful tools for modeling physiological processes, disease progression, and drug responses under controlled microenvironmental conditions. These technologies rely on diverse cell culture methodologies, including 2D and 3D culture formats, spheroids, scaffold-based systems, hydrogels, and organoid models, to recapitulate tissue-level functions and generate rich, multiparametric datasets through high-resolution imaging, integrated sensors, and biochemical assays. The heterogeneity and volume of these data introduce substantial challenges in pre-processing, feature extraction, multimodal integration, and biological interpretation. Artificial intelligence (AI), particularly machine learning and deep learning, offers solutions to these analytical bottlenecks by enabling automated phenotyping, predictive modeling, and real-time control of microfluidic environments. Recent advances also highlight the importance of technical frameworks such as dimensionality reduction, explainable feature selection, spectral pre-processing, lightweight on-chip inference models, and privacy-preserving approaches that support robust and deployable AI–microfluidic workflows. AI-enabled microfluidic and organ-on-a-chip systems now span a broad application spectrum, including cancer biology, drug screening, toxicity testing, microbial and environmental monitoring, pathogen detection, angiogenesis studies, nerve-on-a-chip models, and exosome-based diagnostics. These platforms also hold increasing potential for precision medicine, where AI can support individualized therapeutic prediction using patient-derived cells and organoids. As the field moves toward more interpretable and autonomous systems, explainable AI will be essential for ensuring transparency, regulatory acceptance, and biological insight. Recent AI-enabled applications in cancer modeling, drug screening, etc., highlight how deep learning can enable precise detection of phenotypic shifts, classify therapeutic responses with high accuracy, and support closed-loop regulation of microfluidic environments. These studies demonstrate that AI can transform microfluidic systems from static culture platforms into adaptive, data-driven experimental tools capable of enhancing assay reproducibility, accelerating drug discovery, and supporting personalized therapeutic decision-making. This narrative review synthesizes current progress, technical challenges, and future opportunities at the intersection of AI, microfluidic cell culture platforms, and advanced organ-on-a-chip systems, highlighting their emerging role in precision health and next-generation biomedical research. Full article

Three-dimensional (3D) cell culture systems require biocompatible carriers that provide both structural support and efficient mass transport. Conventional alginate-based encapsulation systems suffer from poor molecular diffusion, lack of cell adhesion motifs, and structural instability under physiological conditions. Here, we report the first development of hollow-shell collagen microcapsules (CMCs) fabricated via a gelatin sacrificial template approach that overcomes these critical limitations. The hollow architecture combined with collagen’s intrinsic bioactivity achieved 2.5-fold enhancement in molecular permeability compared to conventional alginate beads, while maintaining structural integrity for 14 days versus 3-day collapse of alginate controls. NIH 3T3 fibroblasts encapsulated within CMCs demonstrated superior proliferation and formed tissue-like multilayered structures with extensive extracellular matrix deposition. This platform represents a significant advance in 3D cell culture technology, providing a biologically functional microenvironment with enhanced mass transport properties for applications in tissue engineering and regenerative medicine. Full article

Temperature-responsive hydrogels are sophisticated stimuli-responsive biomaterials that undergo rapid, reversible sol–gel phase transitions in response to subtle thermal stimuli, most notably around physiological temperature. This inherent thermosensitivity enables non-invasive, precise spatiotemporal control of material properties and bioactive payload release, rendering them highly promising for advanced biomedical applications. This review critically surveys recent advances in the design, synthesis, and translational potential of thermo-responsive hydrogels, emphasizing nanoscale and hybrid architectures optimized for superior tunability and biological performance. Foundational systems remain dominated by poly(N-isopropylacrylamide) (PNIPAAm), which exhibits a sharp lower critical solution temperature near 32 °C, alongside Pluronic/Poloxamer triblock copolymers and thermosensitive cellulose derivatives. Contemporary developments increasingly exploit biohybrid and nanocomposite strategies that incorporate natural polymers such as chitosan, gelatin, or hyaluronic acid with synthetic thermo-responsive segments, yielding materials with markedly enhanced mechanical robustness, biocompatibility, and physiologically relevant transition behavior. Cross-linking methodologies—encompassing covalent chemical approaches, dynamic physical interactions, and radiation-induced polymerization are rigorously assessed for their effects on network topology, swelling/deswelling kinetics, pore structure, and degradation characteristics. Prominent applications include on-demand drug and gene delivery, injectable in situ gelling systems, three-dimensional matrices for cell encapsulation and organoid culture, tissue engineering scaffolds, self-healing wound dressings, and responsive biosensing platforms. The integration of multi-stimuli orthogonality, nanotechnology, and artificial intelligence-guided materials discovery is anticipated to deliver fully programmable, patient-specific hydrogels, establishing them as pivotal enabling technologies in precision and regenerative medicine. Full article

Extracellular vesicles (EVs) derived from stem cells have emerged as promising mediators of osteogenesis, suggesting cell-free alternatives for bone tissue engineering and regenerative medicine. This review provides a comprehensive analysis of the main stem cell sources used for EV production, including bone marrow mesenchymal stem cells (BM-MSCs), adipose-derived stem cells (ADSCs), umbilical cord MSCs (UC-MSCs), induced pluripotent stem cells (iPSCs), and alternative stromal populations. Particular attention is given to the ways in which different conditioning and differentiation strategies, such as osteogenic induction, hypoxia, and mechanical stimulation, modulate EV cargo composition and enhance their therapeutic potential. We further discuss the in vitro models employed to evaluate EV-mediated bone regeneration, ranging from 2D cultures to complex 3D spheroids, scaffold-based systems, and bone organoids. Overall, this review emphasizes the current challenges related to standardization, scalable production, and clinical translation. It also outlines future directions, including bioengineering approaches, advanced preclinical models, and the integration of multi-omics approaches and artificial intelligence to optimize EV-based therapies. By integrating current knowledge, this work aims to guide researchers toward more consistent and physiologically relevant strategies to harness EVs for effective bone regeneration. Finally, this work uniquely integrates a comparative analysis of EVs from multiple stem cell sources with engineering strategies and emerging clinical perspectives, thereby providing an updated and translational framework for their application in bone regeneration. Full article

Biomimetic scaffolds are required in tissue engineering to provide structural support as well as promote cell adhesion, proliferation, and differentiation. Fibrous scaffolds composed of micro- and nanofibers replicate the architecture of the native extracellular matrix. Electrospinning is widely used for fabricating nanofibers; however, constructing fibrous scaffolds that integrate multiple fiber scales into a single structure is difficult. We addressed this issue by developing a fiber-spinning device using two pairs of counter-facing syringes that simultaneously produce micro- and nanofibers under different processing conditions. Poly(ε-caprolactone) solutions are ejected through needle-type nozzles via centrifugal force, and fiber diameter is controlled by adjusting the polymer concentration and nozzle diameter. We fabricated scaffolds with the proposed device, which exhibited a random three-dimensional fibrous network in which microfibers and nanofibers were homogeneously integrated. C2C12 myoblasts cultured on the composite scaffolds strongly adhered to the fibrous network, remained viable, and extended along the fibers to form multinucleated cells within the structure. The developed system produced composite micro/nanofiber scaffolds with tunable morphology and biocompatibility, providing a platform for fibrous tissue engineering applications. Full article

In this study, an innovative bioreactor, named BioAxFlow, particularly suitable for tissue engineering applications, is tested. Unlike traditional bioreactors, it does not rely on mechanical components to agitate the culture medium, but on the unique fluid-dynamics behaviour induced by the geometry of the culture chamber, which ensures continuous movement of the medium, promoting the constant exposure of the cells to nutrients and growth factors. Using the human osteosarcoma cell line SAOS-2, the bioreactor’s ability to enhance cell adhesion and proliferation on polylactic acid (PLA) scaffolds, mimicking bone matrix architecture, is investigated. Cells cultured in the bioreactor showed significant improvement in cell growth and adhesion, compared to static cultures, and a more homogeneous cell distribution upon the scaffold surfaces, which is crucial for the development of functional tissue constructs. The bioreactor also preserves the osteogenic potential of SAOS-2 cells as assessed by the expression of key osteogenic markers. Additionally, it retains the tumorigenic characteristics of SAOS-2 cells, including the expression of pro-angiogenic factors and apoptosis-related genes. These results indicate that the BioAxFlow bioreactor could be an effective platform for tissue engineering and cancer research, offering a promising tool for both regenerative medicine applications and drug testing. Full article

Calcium phosphate cements (CPCs) and biomaterials, such as mesoporous bioactive glass (MBG), are critical for bone tissue engineering. This study aimed to 3D-print CPC scaffolds modified with MBG to enhance their osteogenic potential and regenerative ability. MBG powder was synthesized and characterized using transmission electron microscopy (TEM), X-ray diffraction (XRD), and nitrogen adsorption–desorption techniques. A commercial CPC ink (hydroxyapatite/α-tricalcium phosphate) was mixed with 5% MBG (w/w; CPC/MBG), and, after rheological assessment, the mixture was used to obtain scaffolds via 3D printing. These scaffolds were then tested for chemical, morphological, and mechanical properties, as well as ion release analysis. Unmodified CPC 3D-printed scaffolds served as controls. Biological experiments, including cell viability, DNA content, cell adhesion/spreading, and osteogenic gene expression, were performed by seeding alveolar bone-derived mesenchymal stem cells onto the scaffolds. Statistics were performed using Student’s t-test and ANOVA with post hoc tests (α = 5%). MBG characterization showed a typical mesoporous structure with aligned microchannels and an amorphous structure. Both formulations released calcium and phosphate ions; however, CPC/MBG also released silicon. Cell viability, adhesion/spreading, and DNA content were significantly greater in CPC/MBG scaffolds compared to CPC (p < 0.05) after 3 and 7 days of culture. Furthermore, CPC/MBG supported increased expression of key osteogenic genes, including collagen (COL1A1), osteocalcin (OCN), and Runt-related transcription factor 2 (RUNX2), after 14 days (p < 0.05). The combination of CPC ink with MBG particles effectively enhances the biocompatibility and osteogenic potential of the scaffold, making it an innovative bioceramic ink formulation for 3D printing personalized scaffolds for bone regeneration. Full article

Bioengineered functional salivary tissues can advance regenerative therapies, preclinical drug testing, and the fundamental understanding of salivary gland dysfunction. Current salivary tissue models are typically Matrigel-based, hydrogel-based or scaffold-free organoid systems, with limited physiological relevance or mimicry of cell-cell and cell-extracellular matrix (ECM) interactions. We previously developed elastin-alginate cryoelectrospun scaffolds (CES) that resemble the topography and viscoelastic properties of healthy salivary ECM, and validated their potential for stromal cell culture, delivery, and in vitro fibrosis modeling. Here, we evaluated the utility of CES to support 3D cocultures of salivary gland epithelial and mesenchymal cells in vitro. We compared CES with honeycomb-like topography (CES-H) to densely packed electrospun nanofibers (NFs) and CES with fibrous topography (CES-F) for their ability to support SIMS epithelial cell attachment, morphology, 3D clustering, phenotype and organization into distinct clusters when cocultured with stromal cells. Both CES-F and CES-H supported epithelial cell attachment and clustering; in particular, CES-H most effectively supported the self-organization of epithelial and stromal cells into distinct 3D clusters resembling the structure of native salivary tissue. Stromal cells were essential for maintaining the phenotype of epithelial cells cultured on CES-H, laying the foundation for the development of in vitro tissue models. Full article

NLRP3 inflammasomes are transient large protein aggregates involved in the regulation of the innate immune response but are also associated with endothelial dysfunction during vascular inflammation. While NLRP3 inflammasome assembly and activation is well characterized in immune cells, its role in endothelial cell function remains incompletely understood. This study analyses the function of promyelocytic leukemia (PML) protein, a nuclear scaffold protein that forms so-called PML nuclear bodies (PML-NBs), in the regulation of NLRP3 inflammasome activation in endothelial cell cultures. Following LPS priming and subsequent ATP-induced activation, PML played a dual role: 1. It enhanced NF-kB-dependent transcription of inflammasome components (NLRP3, pro-caspase-1 and pro-IL-1β). 2. At the same time, a post-translational reduction in NLRP3 protein levels and reduced ASC oligomerization were observed, leading to impaired inflammasome activation, as evidenced by lower caspase-1 activity and reduced IL-1β secretion. Proper formation of PML-NBs was critical for this regulatory effect on NLRP3 inflammasome formation, as PML-NBs retained ASC in the nucleus and post-translationally modified NLRP3, presumably affecting its stability. Taken together, these findings suggest that PML represents a regulatory checkpoint in endothelial inflammasome activation, preventing excessive inflammatory responses that could contribute to vascular dysfunction associated with chronic inflammation. Full article

Cultured meat has progressed from early in vitro cell culture concepts to regulatory approvals and preliminary commercialization, with recent advancements propelled by interdisciplinary innovations in cell line engineering, serum-free media, bioreactor design, and three-dimensional (3D) assembly technologies. This review synthesizes recent developments from 2023 to 2025, utilizing peer-reviewed publications, patent analyses, regulatory frameworks, and media reports to assess global preparedness for large-scale production. Asia has emerged as a leading hub, with China, Japan, South Korea, and Singapore focusing on scaffold-based 3D cultures, bioinks, and serum-free strategies, complemented by national centers and pilot facilities. The United States leverages its technological advancements and established regulatory framework, as evidenced by recent Food and Drug Administration and United States Department of Agriculture approvals. However, potential complications related to political regional bans and legislation may arise. Europe and the UK prioritize defined media, cell optimization, and structured novel-food regulations, with early commercialization primarily in pet food. Looking ahead, the industrialization of cultured meat is anticipated to be driven by process engineering and hybrid product strategies, with initial pilot-to-demonstration facilities established in countries open to alternative food products. Premium and hybrid cultured meat products are expected to enter the market first, while whole-cut cultured meat is likely to remain a premium offering into the early 2030s. Full article