Advantages of Using 3D Spheroid Culture Systems in Toxicological and Pharmacological Assessment for Osteogenesis Research.

Bone differentiation is crucial for skeletal development and maintenance. Its dysfunction can cause various pathological conditions such as rickets, osteoporosis, osteogenesis imperfecta, or Paget's disease. Although traditional two-dimensional cell culture systems have contributed significantly to our understanding of bone biology, they fail to replicate the intricate biotic environment of bone tissue. Three-dimensional (3D) spheroid cell cultures have gained widespread popularity for addressing bone defects. This review highlights the advantages of employing 3D culture systems to investigate bone differentiation. It highlights their capacity to mimic the complex in vivo environment and crucial cellular interactions pivotal to bone homeostasis. The exploration of 3D culture models in bone research offers enhanced physiological relevance, improved predictive capabilities, and reduced reliance on animal models, which have contributed to the advancement of safer and more effective strategies for drug development. Studies have highlighted the transformative potential of 3D culture systems for expanding our understanding of bone biology and developing targeted therapeutic interventions for bone-related disorders. This review explores how 3D culture systems have demonstrated promise in unraveling the intricate mechanisms governing bone homeostasis and responses to pharmacological agents.

A novel and cost-effective method for high-throughput 3D culturing and rhythmic assessment of hiPSC-derived cardiomyocytes using retroreflective Janus microparticles.

BACKGROUND: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) gain attention as a potent cell source in regenerative medicine and drug discovery. With the necessity of the demands for experimental models to create a more physiologically relevant model of the heart in vitro we herein investigate a 3D culturing platform and a method for assessing rhythm in hiPSC-CMs. METHODS: The 3D cell culture PAMCELL™ plate is designed to enable cells to attach exclusively to adhesive patterned areas. These cell adhesive zones, named as micro-patterned pads, feature micron silica beads that are surface-modified with the well-known arginyl-glycyl-aspartic acid (RGD) peptide. RGD binding to the surface of hiPSC-CMs facilitates cell-cell attachment and the formation of uniform-size spheroids, which is controlled by the diameter of the micro-patterned pads. The assessment and evaluation of 3D hiPSC-CMs beating pattern are carried out using reflective properties of retroreflective Janus micro-particle (RJP). These RJPs are modified with an antibody targeting the gap junction protein found on the surface of hiPSC-CM spheroids. The signal assessment system comprises a camera attached to an optical microscope and a white light source. RESULTS: The 3D PAMCELL™ R100 culture plate efficiently generate approximately 350 uniform-sized hiPSC-CM spheroids in each well of a 96-well plate and supported a 20-day culture. Analysis of genes and protein expression levels reveal that iPSC-CM spheroids grown on PAMCELL™ R100 retain cardiac stem cell characteristics and functions, outperforming traditional 2D culture platform. Additionally, the RJPs enable monitoring and evaluation of in vitro beating properties of cardiomyocytes without using complex monitoring setup. The system demonstrates its capability to identify alteration in the rhythmic activity of cardiac cells when exposed to ion channel blockers, nifedipine and E4031. CONCLUSIONS: The integration of the 3D culture method and RJPs in this study establishes a platform for evaluating the rhythmic properties of 3D hiPSC-CMs. This approach holds significant potential for identifying arrhythmias or other cardiac abnormalities, ultimately contributing to the development of more effective therapies for heart diseases.

Enhancement of anti-inflammatory and immunomodulatory effects of adipose-derived human mesenchymal stem cells by making uniform spheroid on the new nano-patterned plates.

Human mesenchymal stem cells (MSCs) are known to have anti-inflammatory and immunomodulatory functions; thus, several MSC products have been applied as cell therapy in clinical trials worldwide. Recent studies have demonstrated that MSC spheroids have superior anti-inflammatory and immunomodulatory functions to a single cell suspension. Current methods to prepare MSC spheroids include hanging drop, concave microwell aggregation, spinner flask, and gravity circulation. However, all these methods have limitations such as low scalability, easy cell clumping, low viability, and irregular size distribution. Here, we present a nano-patterned culture plasticware named PAMcell™ 3D plate to overcome these limitations. Nano-sized silica particles (700 nm) coated with RGD peptide were arrayed into fusiform onto the PLGA film. This uniform array enabled the seeded MSCs to grow only on the silica particles, forming uniform-sized semi-spheroids within 48 h. These MSC spheroids have been shown to have enhanced stemness, anti-inflammatory, and immunomodulatory functions, as revealed by the increased expression of stem cell markers (Oct4, Sox2, and Nanog), anti-inflammatory (IL-10, TSG6, and IDO), and immunomodulatory molecules (HGF, VEGF, CXCR4) both at mRNA and protein expression levels. Furthermore, these MSC spheroids demonstrated an increased palliative effect on glycemic control in a multiple low-dose streptozotocin-induced diabetes model compared with the same number of MSC single cell suspensions. Taken together, this study presents a new method to produce uniform-sized MSC spheroids with enhanced anti-inflammatory and immunomodulatory functions in vitro and in vivo.

Behavior of Muscle-Derived Stem Cells on Silica Nanostructured Substrates.

The aim of the present work was to evaluate the responses of rat muscle-derived stem cells (rMDSCs) to growth on silica nanostructured substrates (SN) with nanoscale topographic surfaces. SN of different sizes (SN-60, SN-150, SN-300, SN-500, and SN-700) were prepared using silica nanoparticles with sizes of 60-700 nm. The prepared SN showed roughness at the nanoscale level. The total number of adherent cells on SN increased with increasing nanoscale level and incubation time. The rMDSCs attached to SN-500 and SN-700 were extensively flattened, whereas those grown on SN-60, SN-150, and SN-300 were more rounded. The rank order of the cell length and height of attached rMDSCs at 5 d on different surfaces was SN-60 ≈ SN-150 >> SN-300 > SN-500 > SN-700 > glass. Compared with rMDSCs grown on SN-60, SN-150, or SN-300, those attached to SN-500 and SN-700 exhibited a distinct morphology with filopodial extensions and stronger expression of focal adhesion, integrin, and actin. An evaluation of the gene expression of adhered rMDSCs showed that rMDSCs grown on SN-300 exhibited a higher environmental stress response than those grown on glass or SN-700. Collectively, our data provide fundamental insight into the cellular response and gene expression of rMDSCs grown on nanostructured substrates.

Development of three-dimensional articular cartilage construct using silica nano-patterned substrate.

Current strategies for cartilage cell therapy are mostly based on the use of autologous chondrocytes. However, these cells have limitations of a small number of cells available and of low chondrogenic ability, respectively. Many studies now suggest that fetal stem cells are more plastic than adult stem cells and can therefore more efficiently differentiate into target tissues. This study introduces, efficiency chondrogenic differentiation of fetal cartilage-derived progenitor cells (FCPCs) to adult cells can be achieved using a three-dimensional (3D) spheroid culture method based on silica nanopatterning techniques. In evaluating the issue of silica nano-particle size (Diameter of 300, 750, 1200 nm), each particle size was coated into the well of a 6-well tissue culture plate. FCPCs (2 x 105 cells/well in 6-well plate) were seeded in each well with chondrogenic medium. In this study, the 300 nm substrate that formed multi-spheroids and the 1200 nm substrate that showed spreading were due to the cell-cell adhesion force(via N-cadherin) and cell-substrate(via Integrin) force, the 750 nm substrate that formed the mass-aggregation can be interpreted as the result of cell monolayer formation through cell-substrate force followed by cell-cell contact force contraction. We conclude that our 3D spheroid culture system contributes to an optimization for efficient differentiation of FCPC, offers insight into the mechanism of efficient differentiation of engineered 3D culture system, and has promise for wide applications in regeneration medicine and drug discovery fields.

Water-soluble mercury ion sensing based on the thymine-Hg2+-thymine base pair using retroreflective Janus particle as an optical signaling probe.

Herein, we report an optical sensing platform for mercury ions (Hg2+) in water based on the integration of Hg2+-mediated thymine-thymine (T-T) stabilization, a biotinylated stem-loop DNA probe, and a streptavidin-modified retroreflective Janus particle (SA-RJP). Two oligonucleotide probes, including a stem-loop DNA probe and an assistant DNA probe, were utilized. In the absence of Hg2+, the assistant DNA probe does not hybridize with the stem-loop probe due to their T-T mismatch, so the surface-immobilized stem-loop DNA probe remains a closed hairpin structure. In the presence of Hg2+, the DNA forms a double-stranded structure with the loop region via Hg2+-mediated T-T stabilization. This DNA hybridization induces stretching of the stem-loop DNA probe, exposing biotin. To translate these Hg2+-mediated structural changes in DNA probe into measurable signal, SA-RJP, an optical signaling label, is applied to recognize the exposed biotin. The number of biospecifically bound SA-RJPs is proportional to the concentration of Hg2+, so that the concentration of Hg2+ can be quantitatively analyzed by counting the number of RJPs. Using the system, a highly selective and sensitive measurement of Hg2+ was accomplished with a limit of detection of 0.027nM. Considering the simplified optical instrumentation required for retroreflection-based RJP counting, RJP-assisted Hg2+ measurement can be accomplished in a much easier and inexpensive manner. Moreover, the detection of Hg2+ in real drinking water samples including tap and commercial bottled water was successfully carried out.

Cultivation of human dermal fibroblasts and epidermal keratinocytes on keratin-coated silica bead substrates.

Human hair keratin is promising as a bioactive material platform for various biomedical applications. To explore its versatility further, human hair keratin was coated onto monolayers of silica beads to produce film-like substrates. This combination was hypothesized to provide a synergistic effect in improving the biochemical properties of the resultant composite. Atomic force microscopy analysis showed uniform coatings of keratin on the silica beads with a slight increase in the resulting surface roughness. Keratin-coated silica beads had higher surface energy and relatively lower negative charge than those of bare silica beads. To investigate cell response, human dermal fibroblasts (HDFs), and human epidermal keratinocytes (HEKs) were cultured on the substrates over 4 days. Results showed that keratin coatings significantly enhanced the metabolic activity of HDFs and encouraged cell spreading but did not exert any significant effects on HEKs. HDF expression of collagen I was significantly more intense on the keratin-coated compared to the bare silica substrates. Furthermore, HDF secretion of various cytokines suggested that keratin coatings triggered active cell responses related to wound healing. Collectively, our study demonstrated that human hair keratin-coated silica bead monolayers have the potential to modulate HDF behavior in culture and may be exploited further. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2789-2798, 2017.

Retroreflective Janus Microparticle as a Nonspectroscopic Optical Immunosensing Probe.

We developed retroreflective Janus microparticles (RJPs) as a novel optical immunosensing probe for use in a nonspectroscopic retroreflection-based immunoassay. By coating the metals on the hemispherical surface of silica particles, highly reflective RJPs were fabricated. On the basis of the retroreflection principle, the RJPs responded to polychromatic white light sources, in contrast to conventional optical probes, which require specific monochromatic light. The retroreflection signals from RJPs were distinctively recognized as shining dots, which can be intuitively counted using a digital camera setup. Using the developed retroreflective immunosensing system, cardiac troponin I, a specific biomarker of acute myocardial infarction, was detected with high sensitivity. On the basis of the demonstrated features of the retroreflective immunosensing platform, we expect that our approach may be applied for various point-of-care-testing applications.

Random networks of single-walled carbon nanotubes promote mesenchymal stem cell's proliferation and differentiation.

Studies on the interaction of cells with single-walled carbon nanotubes (SWCNTs) have been receiving increasing attention owing to their potential for various cellular applications. In this report, we investigated the interactions between biological cells and nanostructured SWCNTs films and focused on how morphological structures of SWCNT films affected cellular behavior such as cell proliferation and differentiation. One directionally aligned SWCNT Langmuir-Blodgett (LB) film and random network SWCNT film were fabricated by LB and vacuum filteration methods, respectively. We demonstrate that our SWCNT LB and network film based scaffolds do not show any cytotoxicity, while on the other hand, these scaffolds promote differentiation property of rat mesenchymal stem cells (rMSCs) when compared with that on conventional tissue culture polystyrene substrates. Especially, the SWCNT network film with average thickness and roughness values of 95 ± 5 and 9.81 nm, respectively, demonstrated faster growth rate and higher cell thickness for rMSCs. These results suggest that systematic manipulation of the thickness, roughness, and directional alignment of SWCNT films would provide the convenient strategy for controlling the growth and maintenance of the differentiation property of stem cells. The SWCNT film could be an alternative culture substrate for various stem cells, which often require close control of the growth and differentiation properties.

Effects of anodized titanium with Arg-Gly-Asp (RGD) peptide immobilized via chemical grafting or physical adsorption on bone cell adhesion and differentiation.

PURPOSE: This study examined the effects of the immobilization of Arg-Gly-Asp (RGD) peptide (CAAALLLKERGDSK) on anodized titanium (Ti) via chemical grafting or physical adsorption methods on cell adhesion and osteoblast differentiation. MATERIALS AND METHODS: The RGD peptide was immobilized on the anodized Ti surface by means of physical adsorption or chemical grafting. The chemical composition of each RGD-immobilized Ti substrate was examined by x-ray photoelectron spectroscopy. The level of cell proliferation was investigated via tetrazolium (XTT) assay. Alkaline phosphatase activity and calcium deposition were evaluated by alizarin red S staining, and mRNA expression of the differentiated osteoblast marker genes was analyzed by reverse-transcriptase polymerase chain reaction. RESULTS: Cell adhesion was enhanced on the RGD-immobilized Ti substrates compared to the anodized Ti surfaces. In addition, significantly increased cell spreading and proliferation were observed with the cells grown on the RGD-immobilized Ti (P < .05). Furthermore, the osteoblasts on the RGD-immobilized Ti showed significant increases in the integrin ?1 and type I collagen levels and small increases in osteonectin and osteocalcin levels (P < .05). Interestingly, the chemical grafting method resulted in significantly greater effects on adhesion and differentiation than the physical adsorption method (P < .05). CONCLUSION: RGD-immobilized Ti substrates might be effective in improving the osseointegration of dental implants. In particular, the chemical grafting method of RGD immobilization is more favorable and is expected to provide positive outcomes with future animal and clinical studies.

Nanostructured films as a novel substrate for chondrocytes growth.

We fabricated a large area silica nano-particle monolayer on glass substrates for the cell growth by the Langmuir-Blodgett technique. A thin film of 300 nm sized mono-dispersed silica particles was constructed on the air-water interface and transferred onto a glass substrate. Chondrocytes were cultured on nano-structured substrates and bare glass substrates for 8 days. The characterizations of chondrocytes on nano-structured substrate were conducted on 3rd and 6th day using confocal laser microscopy and with MTT assay for 8 days. The chondrocytes cultured on nano-structured substrate showed podia like spike and their size was larger than that formed on bare glass substrate. The metabolic activity of chondrocytes on nano-structured substrate was lower than that on bare glass substrate at early-stage, but it was recovered after 4 days.

Fabrication of nano-structured SPR chip for sensitivity enhancement.

We used the Langmuir-Blodgett (LB) method for preparation of large area, defect-free monolayer silica particle film as a template for the fabrication of nano-structure Au pattern on a conventional Au substrate for Surface Plasmon Resonance (SPR). Well organized, trigonal pyramid shaped Au nano-structures were able to construct on 34 separate chips in one fabrication process. The dimensions of trigonal pyramid nano-structures were precisely controlled by changing the particle size of the silica LB template. The nano-structure Au patterned SPR chips provides the enhancement of sensitivity in SPR analysis. The sensitivity enhancement of nano-structure patterned chips was evaluated by comparison with conventional flat Au chips. The nano-structure patterned substrate demonstrated a sensitivity enhancement up to 120% compared to a conventional SPR chip when ethanol solution was used as an analyte.

Hexagonally close packed Langmuir-Blodgett films from monodispersed silica nanoparticles.

This report demonstrates a novel procedure to produce hexagonally close packed nanoparticle film using the Langmuir-Blodgett (LB) technique. The LB technique has advantages over previously reported techniques, such as high reproducibility, formation of large area films with low-cost capable of producing a large number of hexagonally close packed nanoparticle films at a single process. In this method, a monolayer of mono-dispersed silica nanoparticles was spread on the surface of water and then transferred it onto a gold sputtered glass substrate or silicon wafer. The silica particles used for fabrication of LB films were synthesized by the modified Stöber method. In present work, we synthesized 300 +/- 5 nm sized silica particles. A large area of hexagonally close packed nanoparticle films fabricated by the LB technique was confirmed by AFM and FE-SEM. Furthermore, we demonstrate fabrication of over 34 hexagonally close packed nanoparticle LB films on glass substrates with area of 18 x 18 mm2 from a single process.

Analysis of wavelength shift versus surface coverage of proteins on nano-porous silicon immunosensor.

The Fabry-Perot fringe pattern is determined by wavelength shifts in interferometric reflectance spectroscopy, which is a function of the refractive index (n) and thickness (l) of the porous silicon (PSi). In this paper, we demonstrated the shifts of wavelength as systematic controlling of adsorbed concentration of a protein on the PSi substrate. In order to correlate the wavelength shift of the Fabry-Perot fringe pattern with the protein adsorption, adsorbed concentrations of avidin (66 kDa) on the PSi substrate were measured by three independent methods: first, by means of bromophenol blue, which is applied to surface-confined avidin; second, with fluorescence intensity measurement of FITC-tagged avidin; and finally by utilizing Time of Flight-Secondary Ion Mass Spectrometry (TOF-SIMS). It was found that the wavelength shift is directly proportional to the surface-adsorbed avidins over the wide range of surface concentration on the PSi substrate, with a rate of 0.1 nm red-shift at avidin concentration change of 1.0 fmol/cm2.

Directed self-assembly of gold binding polypeptide-protein A fusion proteins for development of gold nanoparticle-based SPR immunosensors.

Effective immobilization of antibodies on a sensing platform and sensitivity enhancement are crucial in designing surface plasmon resonance (SPR) immunosensors. Colloidal gold nanoparticles (AuNPs) were directly assembled onto a surface of SPR Au chip via 2-aminoethanethiol for the enhancement of sensitivity as a label-free detection system. SEM image showed most AuNPs were uniformly distributed over the surface. A novel fusion protein was constructed by genetically fusing gold binding polypeptides (GBP) to protein A (ProA) as a crosslinker for effective immobilization of antibodies. The resulting GBP-ProA protein was directly self-immobilized onto both bare and AuNPs-assembled SPR chip surfaces via the GBP portion, followed by the oriented binding of human immunoglobulin G (hIgG) onto the ProA domain targeting the Fc region of antibodies and anti-hIgG in series. Furthermore, anti-Salmonella antibodies were immobilized onto both GBP-ProA layered chips for detection of Salmonella typhimurium. SPR analyses indicated the signal increases for successive binding of hIgG and anti-hIgG onto the GBP-ProA layered AuNPs-assembled chip were higher (about 92 and 30%, respectively) than that onto the identically treated bare chip. This signal enhancement in the AuNPs-assembled chip also caused a 10-fold increased sensitivity in detection of S. typhimurium compared to the bare one. These results demonstrate the direct assembly of AuNPs onto a SPR chip could enhance the signal in biomolecular interaction events, and the GBP-ProA protein could be a valuable crosslinker for simple and oriented immobilization of antibodies onto Au chip surfaces without any surface chemical modification.

Sensitivity enhancement by Au nanoparticles in surface plasmon resonance chemical sensors.

We investigated the sensitivity enhancement in chemical sensors by coupling Au nanoparticles that have the specific size and surface density on sensor chips as those found in label-free detection systems. The Au particles with 10, 30, 60 nm diameter were conjugated by amine groups of cystamine modified chips. The surface density of Au particles was controlled by the reaction time and the concentration of the solution containing the particles. In order to investigate the sensitivity enhancement, we compared the resonance angle shifts with or without Au particles in a methanol aqueous solution. We know that the sensitivity depends on the size and surface density of particles. The sensitivity increased by 57% with the adsorption of 30 nm diameter particles and low surface density due to the coupling effect of localized surface plasmon derived by the size and density of specific Au nanoparticles and surface plasmon waves.