The development of an efficient platform for the growth and neuronal differentiation of stem cells is crucial for autologous cell therapy and tissue engineering to treat various neuronal disorders and neurodegenerative diseases. In this study, we describe the use of highly uniform graphene platforms that provide unique environments where unusual three-dimensional spheroids of human mesenchymal stem cells (hMSCs) are formed, which is advantageous for the differentiation of hMSCs into neurons. We suppose that graphene regulates the interactions at cell-substrate or cell–cell interfaces, consequently promoting the neurogenesis of hMSCs as well as the outgrowth of neurites, which was evidenced by the graphene-induced upregulation of early neurogenesis-related genes. We also demonstrated that the differentiated neurons from hMSCs on graphene are notably sensitive to external ion stimulation, and their neuronal properties can be maintained even after detaching and re-seeding onto a normal cell culture substrate, suggesting the enhanced maturity of resulting neuronal cells. Thus, we conclude that monolayer graphene is capable of regulating the growth and neural differentiation of hMSCs, which would provide new insight and strategy not only for autologous stem cell therapy but for tissue engineering and regenerative medicine based on graphene scaffolds.

The 3D-printed bioactive ceramic incorporated Poly(ε-caprolactone) (PCL) scaffolds show great promise as synthetic bone graft substitutes. However, 3D-printed scaffolds still lack adequate surface properties for cells to be attached to them. In this study, we modified the surface characteristics of 3D-printed poly(ε-caprolactone)/hydroxyapatite scaffolds using O2 plasma and sodium hydroxide. The surface property of the alkaline hydrolyzed and O2 plasma-treated PCL/HA scaffolds were evaluated using field-emission scanning microscopy (FE-SEM), Alizarin Red S (ARS) staining, and water contact angle analysis, respectively. The in vitro behavior of the scaffolds was investigated using human dental pulp-derived stem cells (hDPSCs). Cell proliferation of hDPSCs on the scaffolds was evaluated via immunocytochemistry (ICC) and water-soluble tetrazolium salt (WST-1) assay. Osteogenic differentiation of hDPSCs on the scaffolds was further investigated using ARS staining and Western blot analysis. The result of this study shows that alkaline treatment is beneficial for exposing hydroxyapatite particles embedded in the scaffolds compared to O2 plasma treatment, which promotes cell proliferation and differentiation of hDPSCs.

Three-dimensional (3D) bioprinting is a technology used to deposit cell-laden biomaterials for the construction of complex tissues. Thermosensitive hydrogels are physically cross-linked by non-covalent interaction without using crosslinkers, facilitating low cytotoxicity and cell viability. Chitosan, which is a non-toxic, biocompatible and biodegradable polysaccharide, can be used as a thermosensitive hydrogel. Therefore, chitosan hydrogel could be of potential use as a 3D bioprinting ink. The purpose of this study was to develop and compare the effectivity of different bioinks based on chitosan hydrogels for 3D bioprinting. The solvent type did not affect the gel shape and gelation time, whereas acetic acid exhibited better biocompatibility compared to lactic and hydrochloric acids. The nature of the gelling agent was found to have a stronger influence on these characteristics than that of the solvent. The NaHCO₃ moiety exhibited a higher growth rate of the storage modulus (G′) and a more irregular porous structure than that of the β-glycerophosphate (β-GP) and K₂HPO₄ groups. Cell viability, and live and dead assays, showed that the NaHCO₃ group was more efficient for cell adhesion. The type of gelling agent did not lead to appreciable differences in cell-laden constructs. The NaHCO₃ group was more amenable to bioprinting, compared to the β-GP and K₂HPO₄ groups. The chitosan hydrogel bioinks could, therefore, be good candidates for 3D bioprinting and would pave the way for patient-specific regenerative medicines.

Stem cells derived from dental tissues—dental stem cells—are favored due to their easy acquisition. Among them, dental pulp stem cells (DPSCs) extracted from the dental pulp have many advantages, such as high proliferation and a highly purified population. Although their ability for neurogenic differentiation has been highlighted and neurogenic differentiation using electrospun nanofibers (NFs) has been performed, graphene-incorporated NFs have never been applied for DPSC neurogenic differentiation. Here, reduced graphene oxide (RGO)-polycaprolactone (PCL) hybrid electrospun NFs were developed and applied for enhanced neurogenesis of DPSCs. First, RGO-PCL NFs were fabricated by electrospinning with incorporation of RGO and alignments, and their chemical and morphological characteristics were evaluated. Furthermore, in vitro NF properties, such as influence on the cellular alignments and cell viability of DPSCs, were also analyzed. The influences of NFs on DPSCs neurogenesis were also analyzed. The results confirmed that an appropriate concentration of RGO promoted better DPSC neurogenesis. Furthermore, the use of random NFs facilitated contiguous junctions of differentiated cells, whereas the use of aligned NFs facilitated an aligned junction of differentiated cells along the direction of NF alignments. Our findings showed that RGO-PCL NFs can be a useful tool for DPSC neurogenesis, which will help regeneration in neurodegenerative and neurodefective diseases.

Cellular behavior is dependent on a variety of physical cues required for normal tissue function. In order to mimic native tissue environments, human alveolar bone-derived mesenchymal stem cells (hABMSCs) were exposed to orbital shear stress (OSS) in a low-speed orbital shaker. The synergistic effects of OSS on proliferation and differentiation of hABMSCs were investigated. In particular, we induced the osteoblastic differentiation of hABMSCs cultured in the absence of OM by exposing hABMSCs to OSS (0.86–1.51 dyne/cm²). Activation of Cx43 was associated with exposure of hABMSCs to OSS. The viability of cells stimulated for 10, 30, 60, 120, and 180 min/day increased by approximately 10% compared with that of control. The OSS groups with stimulation of 10, 30, and 60 min/day had more intense mineralized nodules compared with the control group. In quantification of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-2 (BMP-2) protein, VEGF protein levels under stimulation for 10, 60, and 180 min/day and BMP-2 levels under stimulation for 60, 120, and 180 min/day were significantly different compared with those of the control. In conclusion, the results indicated that exposing hABMSCs to OSS enhanced their differentiation and maturation.

This study was performed to investigate the effects of extremely low frequency pulsed electromagnetic fields (ELF-PEMFs) on the proliferation and differentiation of human alveolar bone-derived mesenchymal stem cells (hABMSCs). Osteogenesis is a complex series of events involving the differentiation of mesenchymal stem cells to generate new bone. In this study, we examined not merely the effect of ELF-PEMFs on cell proliferation, alkaline phosphatase (ALP) activity, and mineralization of the extracellular matrix but vinculin, vimentin, and calmodulin (CaM) expressions in hABMSCs during osteogenic differentiation. Exposure of hABMSCs to ELF-PEMFs increased proliferation by 15% compared to untreated cells at day 5. In addition, exposure to ELF-PEMFs significantly increased ALP expression during the early stages of osteogenesis and substantially enhanced mineralization near the midpoint of osteogenesis within 2 weeks. ELF-PEMFs also increased vinculin, vimentin, and CaM expressions, compared to control. In particular, CaM indicated that ELF-PEMFs significantly altered the expression of osteogenesis-related genes. The results indicated that ELF-PEMFs could enhance early cell proliferation in hABMSCs-mediated osteogenesis and accelerate the osteogenesis.