Nanofiber-microwell cell culture system for spatially patterned differentiation of pluripotent stem cells in 3D.

The intricate interplay between biochemical and physical cues dictates pluripotent stem cell (PSC) differentiation to form various tissues. While biochemical modulation has been extensively studied, the role of biophysical microenvironments in early lineage commitment remains elusive. Here, we introduce a novel 3D cell culture system combining electrospun nanofibers with microfabricated polydimethylsiloxane (PDMS) patterns. This system enables the controlled formation of semispherical human induced pluripotent stem cell (hiPSC) colonies, facilitating the investigation of local mechanical stem cell niches on mechano-responsive signaling and lineage specification. Our system unveiled spatially organized RhoA activity coupled with actin-myosin cable formation, suggesting mechano-dependent hiPSC behaviors. Nodal network analysis of RNA-seq data revealed RhoA downstream regulation of YAP signaling, DNA histone modifications, and patterned germ layer specification. Notably, altering colony morphology through controlled PDMS microwell shaping effectively modulated the spatial distribution of mechano-sensitive mediators and subsequent differentiation. This study provides a cell culture platform to decipher the role of biophysical cues in early embryogenesis, offering valuable insights for material design in tissue engineering and regenerative medicine applications.

Engineering Magnetic Nanoclusters for Highly Efficient Heating in Radio-Frequency Nanowarming.

Effective thawing of cryopreserved samples requires rapid and uniform heating. This is achievable through nanowarming, an approach that heats magnetic nanoparticles by using alternating magnetic fields. Here we demonstrate the synthesis and surface modification of magnetic nanoclusters for efficient nanowarming. Magnetite (Fe3O4) nanoclusters with an optimal diameter of 58 nm exhibit a high specific absorption rate of 1499 W/g Fe under an alternating magnetic field at 43 kA/m and 413 kHz, more than twice that of commercial iron oxide cores used in prior nanowarming studies. Surface modification with a permeable resorcinol-formaldehyde resin (RFR) polymer layer significantly enhances their colloidal stability in complex cryoprotective solutions, while maintaining their excellent heating capacity. The Fe3O4@RFR nanoparticles achieved a high average heating rate of 175 °C/min in cryopreserved samples at a concentration of 10 mg Fe/mL and were successfully applied in nanowarming porcine iliac arteries, highlighting their potential for enhancing the efficacy of cryopreservation.

Enhanced peripheral nerve regeneration by mechano-electrical stimulation.

To address limitations in current approaches for treating large peripheral nerve defects, the presented study evaluated the feasibility of functional material-mediated physical stimuli on peripheral nerve regeneration. Electrospun piezoelectric poly(vinylidene fluoride-trifluoroethylene) nanofibers were utilized to deliver mechanical actuation-activated electrical stimulation to nerve cells/tissues in a non-invasive manner. Using morphologically and piezoelectrically optimized nanofibers for neurite extension and Schwann cell maturation based on in vitro experiments, piezoelectric nerve conduits were synthesized and implanted in a rat sciatic nerve transection model to bridge a critical-sized sciatic nerve defect (15 mm). A therapeutic shockwave system was utilized to periodically activate the piezoelectric effect of the implanted nerve conduit on demand. The piezoelectric nerve conduit-mediated mechano-electrical stimulation (MES) induced enhanced peripheral nerve regeneration, resulting in full axon reconnection with myelin regeneration from the proximal to the distal ends over the critical-sized nerve gap. In comparison, a control group, in which the implanted piezoelectric conduits were not activated in vivo, failed to exhibit such nerve regeneration. In addition, at both proximal and distal ends of the implanted conduits, a decreased number of damaged myelination (ovoids), an increased number of myelinated nerves, and a larger axonal diameter were observed under the MES condition as compared to the control condition. Furthermore, unlike the control group, the MES condition exhibited a superior functional nerve recovery, assessed by walking track analysis and polarization-sensitive optical coherence tomography, demonstrating the significant potential of the piezoelectric conduit-based physical stimulation approach for the treatment of peripheral nerve injury.

Non-destructive characterization of bone mineral content by machine learning-assisted electrochemical impedance spectroscopy.

Continuous quantitative monitoring of the change in mineral content during the bone healing process is crucial for efficient clinical treatment. Current radiography-based modalities, however, pose various technological, medical, and economical challenges such as low sensitivity, radiation exposure risk, and high cost/instrument accessibility. In this regard, an analytical approach utilizing electrochemical impedance spectroscopy (EIS) assisted by machine learning algorithms is developed to quantitatively characterize the physico-electrochemical properties of the bone, in response to the changes in the bone mineral contents. The system is designed and validated following the process of impedance data measurement, equivalent circuit model designing, machine learning algorithm optimization, and data training and testing. Overall, the systematic machine learning-based classification utilizing the combination of EIS measurements and electrical circuit modeling offers a means to accurately monitor the status of the bone healing process.

Development and Utilization of Multifunctional Polymeric Scaffolds for the Regulation of Physical Cellular Microenvironments.

Polymeric biomaterials exhibit excellent physicochemical characteristics as a scaffold for cell and tissue engineering applications. Chemical modification of the polymers has been the primary mode of functionalization to enhance biocompatibility and regulate cellular behaviors such as cell adhesion, proliferation, differentiation, and maturation. Due to the complexity of the in vivo cellular microenvironments, however, chemical functionalization alone is usually insufficient to develop functionally mature cells/tissues. Therefore, the multifunctional polymeric scaffolds that enable electrical, mechanical, and/or magnetic stimulation to the cells, have gained research interest in the past decade. Such multifunctional scaffolds are often combined with exogenous stimuli to further enhance the tissue and cell behaviors by dynamically controlling the microenvironments of the cells. Significantly improved cell proliferation and differentiation, as well as tissue functionalities, are frequently observed by applying extrinsic physical stimuli on functional polymeric scaffold systems. In this regard, the present paper discusses the current state-of-the-art functionalized polymeric scaffolds, with an emphasis on electrospun fibers, that modulate the physical cell niche to direct cellular behaviors and subsequent functional tissue development. We will also highlight the incorporation of the extrinsic stimuli to augment or activate the functionalized polymeric scaffold system to dynamically stimulate the cells.

Magneto- and opto-stimuli responsive nanofibers as a controlled drug delivery system.

The drawbacks of conventional drug administration include repeated administration, non-specific biodistribution in the body's systems, the long-term unsustainability of drug molecules, and high global cytotoxicity, posing a challenge for the efficient treatment of chronic diseases that require varying drug dosages over time for optimal therapeutic efficacy. Most controlled-release methods encapsulate drug molecules in biodegradable materials that dissolve over time to release the drug, making it difficult to deliver drugs on a schedule. To address these limitations, we developed a magneto-, opto-stimuli responsive drug delivery system based on functionalized electrospun nanofibers loaded with superparamagnetic iron oxide nanoparticles (SPIONs). We exploited the Néel relaxation effect of SPIONs, where heat generated from vibrating SPIONs under exogenously applied magnetic fields or laser illumination induced structural changes of the thermo-sensitive nanofibers that encapsulate the particles. We showed that this structural change of nanofibers is the governing factor in controlling the release of dye molecules, used as a model drug and co-encapsulated within the nanofibers. We also showed that the degree of nanofiber structural change depends on SPION loading and duration of stimulation, demonstrating the tunability of the drug release profile. Overall, we demonstrated the potential of SPION-embedded thermoplastic nanofibers as an attractive platform for on-demand drug delivery.

Formation of 3D Self-Organized Neuron-Glial Interface Derived from Neural Stem Cells via Mechano-Electrical Stimulation.

Due to dissimilarities in genetics and metabolism, current animal models cannot accurately depict human neurological diseases. To develop patient-specific in vitro neural models, a functional material-based technology that offers multi-potent stimuli for enhanced neural tissue development is devised. An electrospun piezoelectric poly(vinylidene fluoride-trifluoroethylene) (P(VDF-TrFE)) nanofibrous scaffold is systematically optimized to maximize its piezoelectric properties while accommodating the cellular behaviors of neural stem cells. Hydro-acoustic actuation is elegantly utilized to remotely activate the piezoelectric effect of P(VDF-TrFE) scaffolds in a physiologically-safe manner for the generation of cell-relevant electric potentials. This mechano-electrical stimulation, which arose from the deflection of the scaffold and its consequent generation of electric charges on the scaffold surface under hydro-acoustic actuation, induces the multi-phenotypic differentiation of neural stem cells simultaneously toward neuronal, oligodendrocytic, and astrocytic phenotypes. As compared to the traditional biochemically-mediated differentiation, the 3D neuron-glial interface induced by the mechano-electrical stimulation results in enhanced interactions among cellular components, leading to superior neural connectivity and functionality. These results demonstrate the potential of piezoelectric material-based technology for developing functional neural tissues in vitro via effective neural stem cell modulation with multi-faceted regenerative stimuli.

Mechano-Responsive Piezoelectric Nanofiber as an On-Demand Drug Delivery Vehicle.

The control over biodistribution and pharmacokinetics is critical to enhance the efficacy and minimize the side effects of therapeutic agents. To address the need for an on-demand drug delivery system for precise control over the release time and the quantity of drugs, we exploited the mechano-responsiveness of piezoelectric poly(vinylidene fluoride-trifluroethylene) (P(VDF-TrFE)) nanofibers for drug delivery applications. The large surface area-to-volume ratio inherent to nanomaterials, together with the transformative piezoelectric properties, allowed us to use the material as an ultrasensitive and mechano-responsive drug delivery platform driven by the direct piezoelectric effect. The intrinsic negative zeta potential of the nanofibers was utilized to electrostatically load cationic drug molecules, where surface potential changes by exogenous mechanical actuation trigger the release of drug molecules. We show that the drug release kinetics of the P(VDF-TrFE) nanofibers depends on the fiber diameter, thus piezoelectric properties. We further demonstrated that the drug release quantity can be tuned by the applied pressure or dose of physiologically safe corporeal shockwaves as a mechanical stimulus in in vitro and ex vivo models. Overall, we demonstrated the utility of piezoelectric electrospun nanofibers for mechano-responsive controlled drug release.

Spatially Regulated Multiphenotypic Differentiation of Stem Cells in 3D via Engineered Mechanical Gradient.

Within the osteochondral interface, cellular and extracellular matrix gradients provide a biomechanical and biochemical niche for homeostatic tissue functions. Postnatal joint loading is critical for the development of such tissue gradients, leading to the formation of functional osteochondral tissues composed of superficial, middle, and deep zones of cartilage, and underlying subchondral bone, in a depth-dependent manner. In this regard, a novel, variable core-shell electrospinning strategy was employed to generate spatially controlled strain gradients within three-dimensional scaffolds under dynamic compressive loading, enabling the local strain-magnitude dependent, multiphenotypic stem cell differentiation. Human mesenchymal stem cells (hMSCs) were cultured in electrospun scaffolds with a linear or biphasic mechanical gradient, which was computationally engineered and experimentally validated. The cell/scaffold constructs were subjected to various magnitudes of dynamic compressive strains in a scaffold depth-dependent manner at a frequency of 1 Hz for 2 h daily for up to 42 days in osteogenic media. Spatially upregulated gene expression of chondrogenic markers (ACAN, COL2A1, PRG4) and glycosaminoglycan deposition was observed in the areas of greater compressive strains. In contrast, osteogenic markers (COL1A1, SPARC, RUNX2) and calcium deposition were downregulated in response to high local compressive strains. Dynamic mechanical analysis showed the maintenance of the engineered mechanical gradients only under dynamic culture conditions, confirming the potent role of biomechanical gradients in developing and maintaining a tissue gradient. These results demonstrate that multiphenotypic differentiation of hMSCs can be controlled by regulating local mechanical microenvironments, providing a novel strategy to recapitulate the gradient structure in osteochondral tissues for successful regeneration of damaged joints in vivo and facile development of interfacial tissue models in vitro.

Graphene-Oxide-Decorated Microporous Polyetheretherketone with Superior Antibacterial Capability and In Vitro Osteogenesis for Orthopedic Implant.

Due to its similar elastic modulus of human bones, polyetheretherketone (PEEK) has been considered as an excellent cytocompatible material. However, the bioinertness, poor osteoconduction, and weak antibacterial activity of PEEK limit its wide applications in clinics. In this study, a facile strategy is developed to prepare graphene oxide (GO) modified sulfonated polyetheretherketone (SPEEK) (GO-SPEEK) through a simple dip-coating method. After detailed characterization, it is found that the GO closely deposits on the surface of PEEK, which is attributed to the π-π stacking interaction between PEEK and GO. Antibacterial tests reveal that the GO-SPEEK exhibits excellent suppression toward Escherichia coli. In vitro cell attachment, growth, differentiation, alkaline phosphatase activity, quantitative real-time polymerase chain reaction analyses, and calcium mineral deposition all illustrate that the GO-SPEEK substrate can significantly accelerate the proliferation and osteogenic differentiation of osteoblast-like MG-63 cells compared with those on PEEK and SPEEK groups. These results suggest that the GO-SPEEK has an improved antibacterial activity and cytocompatibility in vitro, showing that the developed GO-SPEEK has a great potential as the bioactive implant material in bone tissue engineering.