Determining Osteogenic Differentiation Efficacy of Pluripotent Stem Cells by Telomerase Activity

BACKGROUND: Bone tissue engineering based on pluripotent stem cells (PSCs) is a new approach to deal with bone defects. Protocols have been developed to generate osteoblasts from PSCs. However, the low efficiency of this process is still an important issue that needs to be resolved. Many studies have aimed to improve efficiency, but developing accurate methods to determine efficacy is also critical. Studies using pluripotency to estimate efficacy are rare. Telomerase is highly associated with pluripotency. METHODS: We have described a quantitative method to measure telomerase activity, telomeric repeat elongation assay based on quartz crystal microbalance (QCM). To investigate whether this method could be used to determine the efficiency of in vitro osteogenic differentiation based on pluripotency, we measured the pluripotency pattern of cultures through stemness gene expression, proliferation ability and telomerase activity, measured by QCM. RESULTS: We showed that the pluripotency pattern determined by QCM was similar to the patterns of proliferation ability and gene expression, which showed a slight upregulation at the late stages, within the context of the general downregulation tendency during differentiation. Additionally, a comprehensive gene expression pattern covering nearly every stage of differentiation was identified. CONCLUSION: Therefore, this assay may be powerful tools for determining the efficiency of differentiation systems based on pluripotency. In this study, we not only introduce a new method for determining efficiency based on pluripotency, but also provide more information about the characteristics of osteogenic differentiation which help facilitate future development of more efficient protocols.

Rapid detection of diethylstilbestrol using a quartz crystal microbalance with gold nanoparticals amplification / 中华预防医学杂志

<p><b>OBJECTIVE</b>To develop a quartz crystal microbalance (QCM) immunosensor with high sensitivity and selectivity for the rapid detection of diethylstilbestrol.</p><p><b>METHODS</b>Dextran was used as reducing agent for preparing gold nanoparticles (AuNPs) with the size of 40 nm. The AuNPs were coupled with anti-DES antibody after amination. A monolayer was generated after immersing the quartz crystal into the solution of 5 mmol/L 11-mercaptoundecanoic acid(MUA) for 16 hours. After the monolayer was activated by 1-ethyl-3-(3-dimethylaminopropry) carbodiimide hydrochloride (EDC·HCl) and N-hydrosuccinimide (NHS), 20 μl of 2.2 mg/ml DES-HS-BSA was dropped onto the surface of crystal to prepare a sensitive membrane which can recognize DES specifically. Then, 50 μl of 1 mol/L ethanolamine (pH 8.5) was used to seal the carboxylic groups to make the sensitive membrane which could identify DES specifically. QCM immunosensor was used as detection platform to optimize the reaction conditions. Under the optimized conditions, 10 μl of 28 μg/ml AuNPs-antibody was mixed with 10 μl of 0.03-2.5 μg/ml DES, and the mixture was added on the sensitive membrane. QCM immunosensor was used to detect the signals and the standard curve was obtained at the same time. The detection limit was calculated based on the standard curve. The specificity was evaluated by testing DES and its analogues with the same concentration.</p><p><b>RESULTS</b>The optimized concentration for the immobilization of DES-HS-BSA on the surface of QCM was 2.2 mg/ml. The optimized concentration for coupling anti-DES antibody with AuNPs was 7 μg/ml and 15 nmol/L, respectively. The optimized concentration of AuNPs-antibody was 14 μg/ml. The logarithm of DES concentration was proportional to the frequency shift in the range of 0.16-500 ng/ml, Δf=-24.170 lgCDES+69.71, R(2)=0.998. The detection limit of this method was 0.13 ng/ml. DES analogues could not influence the detection of DES obviously, so the sensor had good specificity.</p><p><b>CONCLUSION</b>The quartz crystal microbalance immunosensor with gold nanoparticals amplification could detect DES sensitively and rapidly.</p>

Effect of ultraviolet-photofunctionalization of titanium on protein adsorption and competition / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To study the adsorption behavior of bovine serum albumin (BSA) and fibrinogen (Fg) and the competition of them on titanium before and after ultraviolet (UV)-photofunctionalization, and to provide the evidence of photofunctionalization on the surface modification of titanium implants.</p><p><b>METHODS</b>Titanium disks and sensors of quartz crystal microbalance-D (QCM-D) were stored and sealed in the dark for 4 weeks before being divided into two groups, namely the UV-treated group and control group. Samples in the UV-treated group were treated with UV rays for 48 hours. Then the Fg adsorbing property of disks in both groups was tested at 1, 12 and 24 h. Protein films of Fg and BSA formed on QCM sensors after 1 h incubation were imaged via atomic force microscopy (AFM). Then with QCM-D, for both surfaces the adsorption of Fg and BSA as well as the competition between them was tested by introducing proteins with different sequences.</p><p><b>RESULTS</b>After being incubated for 1 and 12 h, UV-treated group attracted more Fg[(0.250 ± 0.005) and (0.172 ± 0.006) mg] than control group did [(0.207 ± 0.004) and (0.144 ± 0.004) mg] (P < 0.05). However, after 24 h incubation, Fg residual on the UV-treated group [(0.080 ± 0.003) mg] was smaller than that in the control group [(0.127 ± 0.004) mg] (P < 0.05). AFM showed protein clustered more densely on UV-treated surfaces than control surfaces and QCM displayed the same result. In addition, when Fg was introduced into QCM-D after BSA, the mass of protein film increased on both surfaces. However, when BSA was introduced after Fg, the mass of protein film on the control group had no change, but slightly decrease on the UV-treated group.</p><p><b>CONCLUSIONS</b>UV-photofunctionalization promotes protein adsorption but has no influence on the competition between Fg and BSA.</p>

Studies on the saliva adsorption and the salivary film property on the hydroxyapatite surface / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To evaluate the thickness and viscoelasticity of whole saliva (WS), parotid saliva (PS) and submandibular/sublingual gland saliva (SMSLS) film adsorption on the hydroxyapatite (HA) surface.</p><p><b>METHODS</b>Ultra-thin layer of HA nanocrystals was coated on the dissipation TiO(2) sensor of gold quartz crystal microbalance using electrophoretic deposition technique. The thickness of the HA layer was measured by the ellipsometer, and element analysis was conducted using X-ray photoelectron spectroscopy. Atomic force microscopy and scanning electron microscope were used to observe its morphology. The in-situ adsorption thickness, the shear elastic modulus and the shear viscosity of salivary layers (WS, PS and SMSLS) on HA surfaces were investigated. The statistical data were analysed by an one-way ANOVA analysis followed by a SNK-q test.</p><p><b>RESULTS</b>The results show that the HA layer was a plate-like morphology with 1.53 ± 0.12 in Ca/P molar ratio, (19.1 ± 0.9) nm in the thickness and (6.5 ± 1.6) nm in the roughness. The thickness of salivary film was SMSLS [(21.84 ± 1.25) nm] > WS[(17.91 ± 1.35) nm] > PS [(14.30 ± 1.03 nm) (P < 0.05). The shear elastic modulus of salivary film was PS [(0.61 ± 0.01) MPa] > SMSLS [(0.31 ± 0.09) MPa] and WS [(0.25 ± 0.03) MPa] (P < 0.05). The trend of the shear viscosity was opposite to one of thickness.</p><p><b>CONCLUSIONS</b>The characteristics of saliva adsorption on HA surface suggest that the thicker, softer and more hydrated properties for the SMSLS and WS films are likely to afford a stronger lubrication to protect oral surfaces from wear and dehydration. The viscoelasticity of the PS film is probably related to the retention covering the oral cavity.</p>