Titanium Surfaces with a Laser-Produced Microchannel Structure Enhance Pre-Osteoblast Proliferation, Maturation, and Extracellular Mineralization In Vitro.

The clinical success of dental titanium implants is profoundly linked to implant stability and osseointegration, which comprises pre-osteoblast proliferation, osteogenic differentiation, and extracellular mineralization. Because of the bio-inert nature of titanium, surface processing using subtractive or additive methods enhances osseointegration ability but limits the benefit due to accompanying surface contamination. By contrast, laser processing methods increase the roughness of the implant surface without contamination. However, the effects of laser-mediated distinct surface structures on the osteointegration level of osteoblasts are controversial. The role of a titanium surface with a laser-mediated microchannel structure in pre-osteoblast maturation remains unclear. This study aimed to elucidate the effect of laser-produced microchannels on pre-osteoblast maturation. Pre-osteoblast human embryonic palatal mesenchymal cells were seeded on a titanium plate treated with grinding (G), sandblasting with large grit and acid etching (SLA), or laser irradiation (L) for 3-18 days. The proliferation and morphology of pre-osteoblasts were evaluated using a Trypan Blue dye exclusion test and fluorescence microscopy. The mRNA expression, protein expression, and protein secretion of osteogenic differentiation markers in pre-osteoblasts were evaluated using reverse transcriptase quantitative polymerase chain reaction, a Western blot assay, and a multiplex assay, respectively. The extracellular calcium precipitation of pre-osteoblast was measured using Alizarin red S staining. Compared to G- and SLA-treated titanium surfaces, the laser-produced microchannel surfaces enhanced pre-osteoblast proliferation, the expression/secretion of osteogenic differentiation markers, and extracellular calcium precipitation. Laser-treated titanium implants may enhance the pre-osteoblast maturation process and provide extra benefits in clinical application.

Investigation of in vitro bioactivity, and osteoblast and angiogenic activity of spray-dried boron-doped 58S bioactive glass microspheres.

Bioactive glass is a potential biomaterial for bone reconstruction owing to its superior bioactivity and non-toxicity. Yet, the absence of a circulatory system to carry waste and nutrients is a key issue with biomaterials implanted in the body. Thus the development of functional and vascularized new tissue requires the development of angiogenesis, which involves the formation of new blood vessels. Based on this perspective, we aimed to fabricate boron-doped 58S bioactive glass microspheres using the spray drying method, which could offer great flowability, controllable morphology, and narrow size distribution. Characterization of particle morphology and elemental composition were examined using scanning electron microscopy along with energy dispersive spectroscopy, respectively. To evaluate the effect of the boron dopant on in vitro bioactivity, X-ray diffraction and Fourier transform infrared spectroscopy were employed, while MC3T3-E1 osteoblast cells and BAOEC endothelial cells were used to assess the in vitro osteoblast and angiogenic activities, respectively. Finally, the results showed that two distinct morphologies, smooth and concave spheres, were found, with discussion of the corresponding formation mechanism. In addition, positive effects of the boron dopant were demonstrated on the in vitro bioactivity, and osteoblast and angiogenic activity when compared to the un-doped BG specimen.

Lab-on-PCB: One step away from the accomplishment of µTAS?

The techniques, protocols, and advancements revolving around printed circuit boards (PCBs) have been gaining sustained attention in the realm of micro-total analysis systems (µTAS) as more and more efforts are devoted to searching for standardized, highly reliable, and industry-friendly solutions for point-of-care diagnostics. In this Perspective, we set out to identify the current state in which the field of µTAS finds itself, the challenges encountered by researchers in the implementation of these technologies, and the potential improvements that can be targeted to meet the current demands. We also line up some trending innovations, such as 3D printing and wearable devices, along with the development of lab-on-PCB to increase the possibility of multifunctional biosensing activities propelled by integrated microfluidic networks for a wider range of applications, anticipating to catalyze the full potential of µTAS.

Microfluidic Determination of Distinct Membrane Transport Properties between Lung Adenocarcinoma Cells CL1-0 and CL1-5.

The cell membrane permeability of a cell type to water (Lp) and cryoprotective agents (Ps), is the key factor that determines the optimal cooling and mass transportation during cryopreservation. The human lung adenocarcinoma cell line, CL1, has been widely used to study the invasive capabilities or drug resistance of lung cancer cells. Therefore, providing accurate databases of the mass transport properties of this specific cell line can be crucial for facilitating either flexible and optimal preservation, or supply. In this study, utilizing our previously proposed noncontact-based micro-vortex system, we focused on comparing the permeability phenomenon between CL1-0 and its more invasive subline, CL1-5, under several different ambient temperatures. Through the assay procedure, the cells of favor were virtually trapped in a hydrodynamic circulation to provide direct inspection using a high-speed camera, and the images were then processed to achieve the observation of a cell's volume change with respect to time, and in turn, the permeability. Based on the noncontact nature of our system, we were able to manifest more accurate results than their contact-based counterparts, excluding errors involved in estimating the cell geometry. As the results in this experiment showed, the transport phenomena in the CL1-0 and CL1-5 cell lines are mainly composed of simple diffusion through the lipid bilayer, except for the case where CL1-5 were suspended in the cryoprotective agent (CPA) solution, which also demonstrated higher Ps values. The deviated behavior of CL1-5 might be a consequence of the altered expression of aquaporins and the coupling of a cryoprotective agent and water, and has given a vision on possible studies over these properties, and their potential relationship to invasiveness and metastatic stability of the CL1 cell line.

The neutrophil elastase-upregulated placenta growth factor promotes the pathogenesis and progression of periodontal disease.

BACKGROUND: Periodontal disease is a chronic inflammatory disease. Given its high prevalence, especially in aging population, the detailed mechanisms about pathogenesis of periodontal disease are important issues for study. Neutrophil firstly infiltrates to periodontal disease-associated pathogen loci and amplifies the inflammatory response for host defense. However, excessive neutrophil-secreted neutrophil elastase (NE) damages the affected gingival. In lung and esophageal epithelium, NE had been proved to upregulate several growth factors including placenta growth factor (PGF). PGF is an angiogenic factor with proinflammatory properties, which mediates the progression of inflammatory disease. Therefore, we hypothesize excessive NE upregulates PGF and participates in the pathogenesis and progression of periodontal disease. METHODS: In gingival epithelial cells (GEC), growth factors array demonstrated NE-increased growth factors and further be corroborated by Western blot assay and ELISA. The GEC inflammation was evaluated by ELISA. In mice, the immunohistochemistry results demonstrated ligature implantation-induced neutrophil infiltration and growth factor upregulation. By multiplex assay, the ligature-induced proinflammatory cytokines level in gingival crevicular fluid (GCF) were evaluated. Finally, alveolar bone absorption was analyzed by micro-CT images and H & E staining. RESULTS: NE upregulated PGF expression and secretion in GEC. PGF promoted GEC to secret IL-1ß, IL-6, and TNF-α in GCF In periodontal disease animal model, ligature implantation triggered NE infiltration and PGF expression. Blockade of PGF attenuated the ligature implantation-induced IL-1ß, IL-6, TNF-α and MIP-2 secretion and ameliorated the alveolar bone loss in mice. CONCLUSION: In conclusion, the NE-induced PGF triggers gingival epithelium inflammation and promotes the pathogenesis and progression of periodontal disease.

The non-contact-based determination of the membrane permeability to water and dimethyl sulfoxide of cells virtually trapped in a self-induced micro-vortex.

The cell-membrane permeabilities of a cell type toward water (Lp) and cryoprotective agents (Ps) provide crucial cellular information for achieving optimal cryopreservation in the biobanking industry. In this work, cell membrane permeability was successfully determined via directly visualizing the transient profile of the cell volume change in response to a sudden osmotic gradient instantaneously applied between the intracellular and extracellular environments. A new micro-vortex system was developed to virtually trap the cells of interest in flow-driven hydrodynamic circulation passively formed at the expansion region in a microfluidic channel, where trapped cells remain in suspension and flow with the streamline of the localized vortex, involving no physical contact between cells and the device structure; furthermore, this supports a pragmatic assumption of 100% sphericity and allows for the calculation of the active surface area of the cell membrane for estimating the actual cell volume from two-dimensional images. For an acute T-cell lymphoma cell line (Jurkat), moderately higher values (Lp = 0.34 µm min-1 atm-1 for a binary system, and Lp = 0.16 µm min-1 atm-1 and Ps = 0.55 × 10-3 cm min-1 for a ternary system) were measured than those obtained from prior methods utilizing contact-based cell-trapping techniques, manifesting the influence of physical contact on accuracy during the determination of cell membrane permeability.

The pursuit of further miniaturization of screen printed micro paper-based analytical devices utilizing controlled penetration towards optimized channel patterning.

One of the main objectives of microfluidic paper-based analytical devices is to present solutions particularly, for applications in low-resource settings. Therefore, screen-printing appears to be an attractive fabrication technique in the field, due to its overall simplicity, affordability, and high-scalability potential. Conversely, the minimum feature size attained using screen-printing is still rather low, especially compared to other fabrication methods, mainly attributed to the over-penetration of hydrophobic agents, underneath defined patterns on masks, into the fiber matrix of paper substrates. In this work, we propose the use of the over-penetration to our advantage, whereby an appropriate combination of hydrophobic agent temperature and substrate thickness, allows for the proper control of channel patterning, rendering considerably higher resolutions than prior arts. The implementation of Xuan paper and nail oil as novel substrate and hydrophobic agent, respectively, is proposed in this work. Under optimum conditions of temperature and substrate thickness, the resolution of the screen-printing method was pushed up to 97.83 ± 16.34 µm of channel width with acceptable repeatability. It was also found that a trade-off exists between achieving considerably high channel resolutions and maintaining high levels of repeatability of the process. Lastly, miniaturized microfluidic channels were successfully patterned on pH strips for colorimetric pH measurement, demonstrating its advantage on negligible sample-volume consumption in nano-liter range during chemical measurement and minimal interference on manipulation of precious samples, which for the first time, is realized on screen-printed microfluidic paper-based analytical devices.

A Microfluidic Study of Megakaryocytes Membrane Transport Properties to Water and Dimethyl Sulfoxide at Suprazero and Subzero Temperatures.

Megakaryocytes (MKs) are the precursor cells of platelets. Cryopreservation of MKs is critical for facilitating research investigations about the biology of this important cell and may help for scaling-up ex-vivo production of platelets from MKs for clinical transfusion. Determining membrane transport properties of MKs to water and cryoprotectant agents (CPAs) is essential for developing optimal conditions for cryopreserving MKs. To obtain these unknown parameters, membrane transport properties of the human UT-7/TPO megakaryocytic cell line were investigated using a microfluidic perfusion system. UT-7/TPO cells were immobilized in a microfluidic system on poly-D-lysine-coated glass substrate and perfused with various hyper-osmotic salt and CPA solutions at suprazero and subzero temperatures. The kinetics of cell volume changes under various extracellular conditions were monitored by a video camera and the information was processed and analyzed using the Kedem-Katchalsky model to determine the membrane transport properties. The osmotically inactive cell volume (V(b)=0.15), the permeability coefficient to water (Lp) at 37°C, 22°C, 12°C, 0°C, -5°C, -10°C, and -20°C, and dimethyl sulfoxide (DMSO; Ps) at 22, 12, 0, -10, -20, as well as associated activation energies of water and DMSO at different temperature regions were obtained. We found that MKs have relatively higher membrane permeability to water (Lp=2.62 µm/min/atm at 22°C) and DMSO (Ps=1.8×10(-3) cm/min at 22°C) than most other common mammalian cell types, such as lymphocytes (Lp=0.46 µm/min/atm at 25°C). This information could suggest a higher optimal cooling rate for MKs cryopreservation. The discontinuity effect was also found on activation energy at 0°C-12°C in the Arrhenius plots of membrane permeability by evaluating the slope of linear regression at each temperature region. This phenomenon may imply the occurrence of cell membrane lipid phase transition.

Size-selective immunofluorescence of Mycobacterium tuberculosis cells by capillary- and viscous forces.

Rapid, low cost screening of tuberculosis requires an effective enrichment method of Mycobacterium tuberculosis (MTB) cells. Currently, microfiltration and centrifugation steps are frequently used for sample preparation, which are cumbersome and time-consuming. In this study, the size-selective capturing mechanism of a microtip-sensor is presented to directly enrich MTB cells from a sample mixture. When a microtip is withdrawn from a spherical suspension in the radial direction, the cells that are concentrated by AC electroosmosis are selectively enriched to the tip due to capillary- and viscous forces. The size-selectivity is characterized by using polystyrene microspheres, which is then applied to size-selective capture of MTB from a sample mixture. Our approach yields a detection limit of 800 cells mL(-1), one of the highest-sensitivity immunosensors to date.