Synergistic Effect of Cell-Derived Extracellular Matrices and Topography on Osteogenesis of Mesenchymal Stem Cells

Cell-derived matrices (CDMs) are an interesting alternative to conventional sources of extracellular matrices (ECMs) as CDMs mimic the natural ECM composition better and are therefore attractive as a scaffolding material for regulating the functions of stem cells. Previous research on stem cell differentiation has demonstrated that both surface topography and CDMs have a significant influence. However, not much focus has been devoted to elucidating possible synergistic effects of CDMs and topography on osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBM-MSCs). In this study, polydimethylsiloxane (PDMS)-based anisotropic topographies (wrinkles) with various topography dimensions were prepared and subsequently combined with native ECMs produced by human fibroblasts that remained on the surface topography after decellularization. The synergistic effect of CDMs combined with topography on osteogenic differentiation of hBM-MSCs was investigated. The results showed that substrates with specific topography dimensions, coated with aligned CDMs, dramatically enhanced the capacity of osteogenesis as investigated using immunofluorescence staining for identifying osteopontin (OPN) and mineralization. Furthermore, the hBM-MSCs on the substrates decorated with CDMs exhibited a higher percentage of (Yes-associated protein) YAP inside the nucleus, stronger cell contractility, and greater formation of focal adhesions, illustrating that enhanced osteogenesis is partly mediated by cellular tension and mechanotransduction following the YAP pathway. Taken together, our findings highlight the importance of ECMs mediating the osteogenic differentiation of stem cells, and the combination of CDMs and topography will be a powerful approach for material-driven osteogenesis

Sono-processes:Emerging systems and their applicability within the (bio-)medical field

Sonochemistry, although established in various fields, is still an emerging field finding new effects of ultrasound on chemical systems and are of particular interest for the biomedical field. This interdisciplinary area of research explores the use of acoustic waves with frequencies ranging from 20 kHz to 1 MHz to induce physical and chemical changes. By subjecting liquids to ultrasonic waves, sonochemistry has demonstrated the ability to accelerate reaction rates, alter chemical reaction pathways, and change physical properties of the system while operating under mild reaction conditions. It has found its way into diverse industries including food processing, pharmaceuticals, material science, and environmental remediation. This review provides an overview of the principles, advancements, and applications of sonochemistry with a particular focus on the domain of (bio-)medicine. Despite the numerous benefits sonochemistry has to offer, most of the research in the (bio-)medical field remains in the laboratory stage. Translation of these systems into clinical practice is complex as parameters used for medical ultrasound are limited and toxic side effects must be minimized in order to meet regulatory approval. However, directing attention towards the applicability of the system in clinical practice from the early stages of research holds significant potential to further amplify the role of sonochemistry in clinical applications.</p

The Relationship between Bulk Silicone and Benzophenone-Initiated Hydrogel Coating Properties

Polydimethylsiloxane (PDMS) is a silicone elastomer-based material that is used in various applications, including coatings, tubing, microfluidics, and medical implants. PDMS has been modified with hydrogel coatings to prevent fouling, which can be done through UV-mediated free radical polymerization using benzophenone. However, to the best of our knowledge, the properties of hydrogel coatings and their influence on the bulk properties of PDMS under various preparation conditions, such as the type and concentration of monomers, and UV treatment time, have never been investigated. Acrylate-based monomers were used to perform free radical polymerization on PDMS surfaces under various reaction conditions. This approach provides insights into the relationship between the hydrogel coating and bulk properties of PDMS. Altering the UV polymerization time and the monomer concentration resulted in different morphologies with different roughness and thickness of the hydrogel coating, as well as differences in the bulk material stiffness. The surface morphology of the coated PDMS was characterized by AFM. The cross section and thickness of the coatings were examined using scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy. The dependence of coating development on the monomer type and concentration used was evaluated by surface hydrophilicity, as measured by water contact angle. Elongation-until-break analysis revealed that specific reaction conditions affected the bulk properties and made the coated PDMS brittle. Therefore, boundary conditions have been identified to enable high quality hydrogel coating formation without affecting the bulk properties of the material

Topography-Mediated Enhancement of Nonviral Gene Delivery in Stem Cells

Gene delivery holds great promise for bioengineering, biomedical applications, biosensors, diagnoses, and gene therapy. In particular, the influence of topography on gene delivery is considered to be an attractive approach due to low toxicity and localized delivery properties. Even though many gene vectors and transfection systems have been developed to enhance transfection potential and combining it with other forms of stimulations could even further enhance it. Topography is an interesting surface property that has been shown to stimulate differentiation, migration, cell morphology, and cell mechanics. Therefore, it is envisioned that topography might also be able to stimulate transfection. In this study, we tested the hypothesis "topography is able to regulate transfection efficiency", for which we used nano- and microwave-like topographical substrates with wavelengths ranging from 500 nm to 25 µm and assessed the transfectability of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and myoblasts. For transfection, Lipofectamine 2000 and a gene encoding plasmid for red-fluorescent protein (m-Cherry) were used and topography-induced cell morphology and transfection efficiency was analyzed. As a result, topography directs cell spreading, elongation, and proliferation as well as the transfection efficiency, which were investigated but were found not to be correlated and dependent on the cell type. A 55% percent improvement of transfection efficiency was identified for hBM-MSCs grown on 2 µm wrinkles (24.3%) as compared to hBM-MSCs cultured on flat controls (15.7%). For myoblast cells, the highest gene-expression efficiency (46.1%) was observed on the 10 µm topography, which enhanced the transfection efficiency by 64% as compared to the flat control (28.1%). From a qualitative assessment, it was observed that the uptake capacity of cationic complexes of TAMRA-labeled oligodeoxynucleotides (ODNs) was not topography-dependent but that the intracellular release was faster, as indicated by the positively stained nuclei on 2 μm for hBM-MSCs and 10 μm for myoblasts. The presented results indicate that topography enhances the gene-delivery capacity and that the responses are dependent on cell type. This study demonstrates the important role of topography on cell stimulation for gene delivery as well as understanding the uptake capacity of lipoplexes and may be useful for developing advanced nonviral gene delivery strategies

Dynamic Covalent Cross-Linked Nanogel-Stabilized Pickering Emulsion for Responsive Microstructures

Designing new dynamic matrices in combination with a highly diverse material formation approach as Pickering emulsions provides the tools to engineer innovative dynamic porous microstructures in a highly controllable fashion. Here, nanogels (nGels) are used, which exhibit dynamic covalent cross-linking capabilities, as surface stabilizing agents in view of their highly controllable physiochemical properties. The method provides the successful formation of dynamic covalent cross-linked hydrogel microstructures based on ketone and amine-functionalized nGels using Pickering emulsions. In this system, a pH-triggerable responsive behavior is incorporated. The physiochemical properties of the resulting microstructure can be further tailored by modifying the intramolecular interactions at the interface, making these systems interesting for a wide range of applications

High-Throughput Screening and Hierarchical Topography-Mediated Neural Differentiation of Mesenchymal Stem Cells

Biophysical factors such as anisotropic topography composed of micro/nanosized structures are important for directing the fate of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and have been applied to neuronal differentiation. Via high-throughput screening (HTS) methods based on topography gradients, the optimum topography is determined and translated toward a hierarchical architecture designed to mimic the nerve nano/microstructure. The polydimethylsiloxane (PDMS)-based topography gradient with amplitudes (A) from 541 to 3073 nm and wavelengths (W) between 4 and 30 µm is developed and the fate commitment of MSC toward neuron lineage is investigated. The hierarchical structures, combining nano- and microtopography (W0.3/W26 parallel/perpendicular) are fabricated to explore the combined topography effects on neuron differentiation. From the immunofluorescent staining results (Tuj1 and MAP2), the substrate characterized by W: 26 µm; A: 2.9 µm shows highest potential for promoting neurogenesis. Furthermore, the hierarchical features (W0.3/W26 parallel) significantly enhance neural differentiation. The hBM-MSCs on the hierarchical substrates exhibit a significantly lower percentage of nuclear Yes-associated protein (YAP)/TAZ and weaker cell contractility indicating that the promoted neurogenesis is mediated by the cell tension and YAP/TAZ pathway. This research provides new insight into designing biomaterials for applications in neural tissue engineering and contributes to the understanding of topography-mediated neuronal differentiation

pH Sensitive Dextran Coated Fluorescent Nanodiamonds as a Biomarker for HeLa Cells Endocytic Pathway and Increased Cellular Uptake

Fluorescent nanodiamonds are a useful for biosensing of intracellular signaling networks or environmental changes (such as temperature, pH or free radical generation). HeLa cells are interesting to study with these nanodiamonds since they are a model cell system that is widely used to study cancer-related diseases. However, they only internalize low numbers of nanodiamond particles very slowly via the endocytosis pathway. In this work, we show that pH-sensitive, dextran-coated fluorescent nanodiamonds can be used to visualise this pathway. Additionally, this coating improved diamond uptake in HeLa cells by 5.3 times (*** p < 0.0001) and decreased the required time for uptake to only 30 min. We demonstrated further that nanodiamonds enter HeLa cells via endolysosomes and are eventually expelled by cells

3D-Printable Hierarchical Nanogel-GelMA Composite Hydrogel System

The strength of the extracellular matrix (ECM) is that it is hierarchical in terms of matrix built-up, matrix density and fiber structure, which allows for hormones, cytokines, and other small biomolecules to be stored within its network. The ECM-like hydrogels that are currently used do not possess this ability, and long-term storage, along with the need for free diffusion of small molecules, are generally incompatible requirements. Nanogels are able to fulfill the additional requirements upon successful integration. Herein, a stable hierarchical nanogel–gelatin methacryloyl (GelMA) composite hydrogel system is provided by covalently embedding nanogels inside the micropore network of GelMA hydrogel to allow a controlled local functionality that is not found in a homogenous GelMA hydrogel. Nanogels have emerged as a powerful tool in nanomedicine and are highly versatile, due to their simplicity of chemical control and biological compatibility. In this study, an N-isopropylacrylamide-based nanogel with primary amine groups on the surface was modified with methacryloyl groups to obtain a photo-cross-linking ability similar to GelMA. The nanogel-GelMA composite hydrogel was formed by mixing the GelMA and the photo-initiator within the nanogel solution through UV irradiation. The morphology of the composite hydrogel was observed by scanning electron microscopy, which clearly showed the nanogel wrapped within the GelMA network and covering the surface of the pore wall. A release experiment was conducted to prove covalent bonding and the stability of the nanogel inside the GelMA hydrogel. In addition, 3D printability studies showed that the nanogel-GelMA composite ink is printable. Therefore, the suggested stable hierarchical nanogel-GelMA composite hydrogel system has great potential to achieve the in situ delivery and controllable release of bioactive molecules in 3D cell culture systems