Detection and quantification of the metabolite Ac-Tß1-14 in in vitro experiments and urine of rats treated with Ac-Tß4: A potential biomarker of Ac-Tß4 for doping tests.

Thymosin ß4 (Tß4) was reported to exert various beneficial bioactivities such as tissue repair, anti-inflammation, and reduced scar formation, and it is listed on the prohibited substances in sports by the World Anti-Doping Agency. However, no metabolism studies of Tß4 were reported yet. Previously, our lab reported in in vitro experiment that a total of 13 metabolites were found by using multiple enzymes, and six metabolites (Ac-Tß31-43 , Ac-Tß17-43 , Ac-Tß1-11 , Ac-Tß1-14 , Ac-Tß1-15 , and Ac-Tß1-17 ) were confirmed by comparing with the synthetic standards. This study was aimed at identifying new metabolites of Tß4 leucine aminopeptidase (LAP), human kidney microsomes (HKM), cultured huvec cells, and rats after administration of Tß4 protein to develop biomarkers for detecting doping drugs in sports. A method for detecting and quantifying Ac-Tß1-14 was developed and validated using Q-Exactive orbitrap mass spectrometry. The limit of detection (LOD) and limit of quantification (LOQ) of the Ac-Tß1-14 were 0.19 and 0.58 ng/mL, respectively, and showed a good linearity (r2 = 0.9998). As a result, among the six metabolites above, Ac-Tß1-14 , as a common metabolite, was found in LAP, HKM, huvec cells exposed to Tß4, and the urine of rats intraperitoneally treated with 20-mg/kg Tß4. And the metabolite Ac-Tß1-14 was quantitatively determined by 48 h in rats, with the highest concentration occurring between 0 and 6 h. Ac-Tß1-14 was not detected in non-treated control groups, including human blank urine. These results suggest that Ac-Tß1-14 in urine is a potential biomarker for screening the parent Tß4 in doping tests.

Endothelial Cell-Derived Tethered Lipid Bilayers Generating Nitric Oxide for Endovascular Implantation.

Engineering an endothelium-mimetic surface has been one of long-lasting topics to develop ideal cardiovascular devices. The aim of the study was to investigate the potential use of a model of lipid bilayers that not only come from membranes extracted from endothelial cells (ECs) but also embedded with a type of organoselenium lipid enabling it to catalyze the generation of nitric oxide (NO). Herein, the titanium-cloaking in lipid bilayers extracted from ECs was prepared to propose a promising idea for the development of endovascular implants. For this purpose, we synthesized and characterized a lipidic molecule containing selenium and verified enough catalytic activity for the NO generation in the presence of S-nitrosothiols (RSNO) as endogenous NO precursors. We demonstrated the fabrication process of tethered lipid bilayers, from membrane extraction to vesicle fusion, and validated the successful formation of the layer and the catalyst insertion. The resulting bilayer presented endothelium-similar properties including the NO generation and cellular interactions. The catalyst inserted into the bilayer provided an unexampled result in the release period and kinetics of NO, likely similar to the native endothelial system. Using three different cells including EC, smooth muscle cell (SMC), and macrophage, it was demonstrated that the membrane responds selectively to each cell in the manner of promotive, suppressive, and nonimmunoreactive, respectively. Taken together, the fundamental study on obtained results not only provides understanding of the kinetics of designed NO catalyst and cellular interactions of reassembled membranes but also suggests very useful data on rational design and development of many vascular implantable devices, even expandable toward to nonvascular biointerfacing devices.

In vitro and in vivo evaluation of bioglass microspheres incorporated brushite cement for bone regeneration.

Bioglass-calcium phosphate cement (CPC) composite materials have recently received increased attention for bone regeneration purposes, owing to their improved properties in term of biocompatibility and bone ingrowths. In this study, an injectable bone substitute (IBS) system which utilizes bioglass microspheres incorporated into brushite based cement, was evaluated. The microspheres were synthesized with a simple and low sintering temperature process; there was no significant phase difference shown from the powder and good interactivity with cells was obtained. Furthermore, physical properties were optimized in microsphere incorporated brushite cement in order to investigate in vitro and in vivo performance. Accordingly, setting time and compressive strength were hardly altered until a microsphere content of 40% (v/v) was reached. The brushite (BR)/bioglass microsphere (BM) system showed excellent bioactivity to the in-vitro simulated body fluid test: dissolution ions from composite materials influenced apatite growth, countered acidic pH, and increased material degradation. In an in-vitro study with preosteoblasts (MC3T3-E1), BR/BM supported cell adhesion and proliferation, while cell differentiation experiments without osteogenic supplements, demonstrated that BR/BM induced osteogenic differentiation. A post-implantation study conducted in femoral defects showed higher materials degradation and bone formation in BR/BM than in BR. The faster dissolution of bioglass microspheres increased BR/BM composite resorption and hence facilitated bone tissue integration. Our findings suggest that bioglass microspheres incorporated in cement could potentially be used as an injectable bone substitute for bone regeneration applications.

Preparation and evaluation of BCP-CSD-agarose composite microsphere for bone tissue engineering.

Composite microspheres have been widely investigated over the years in order to achieve a sound scaffold with suitable combinations of biodegradable polymers and bioactive ceramics/glasses for bone tissue engineering. In our present study, composite microspheres were prepared for the first time by agarose (1 wt %) enforcement with combination of biphasic calcium phosphate (BCP; 20 wt %) and calcium sulfate dehydrate (CSD; 20 wt %), and analyzed for use in bone regeneration. The one-step fabrication process revealed spheres of sizes ranging from 50 to 1000 µm of BCP-CSD contents effectively formed by natural solidification of agarose matrix, which is very simple, time and cost-effective, and could allow for large scale production. Furthermore, the BCP-CSD-agarose composite microspheres were tested in in vitro and in vivo for bone-forming properties in order to assess their biocompatibility. The rapid diffusion of Ca 2+ ions from CSD of the composite microspheres through agarose matrix potentially increased interactivity with microenvironment and gave support for cell adhesion and proliferation. Moreover, in vivo result demonstrated that fabricated microspheres promoted neovascularization, stimulated fibroblast cell proliferation, and host cell migration occurred throughout the defects and within microspheres, ultimately guided to new bone formation. The developed composite microspheres with novel approach could have potential for bone regeneration application. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2263-2272, 2019.

Effects of platelet-rich plasma on biological activity and bone regeneration of brushite-based calcium phosphate cement.

In this study, we focused on identifying the effects of platelet-rich plasma (PRP) on bioactivity and bone ingrowths incorporated into brushite cement. We introduced PRP as a series of substitutions with an aqueous citrate-ion solution, and the optimized cement with PRP showed no disintegration of the paste consistency. Incorporating PRP showed that the setting time decreased with the increasing of PRP ratios, although the compressive strength was not significantly changed. We evaluated in vitro degradation and bioactivity with the simulated body fluid test, and the result showed that adding PRP accelerated the carbonated apatite nucleation and markedly improved the surface reactivity of the cement. The in vitro studies demonstrated that incorporating PRP into the brushite cement improved cell adhesion and proliferation. The in vivo effects of PRP were faster degradation, improved tissue response in the early stage, and bone ingrowths. We demonstrated based on our results of this study, incorporation of PRP into brushite cement could be helpful for improving biological activity of the cement as well as bone regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2316-2326, 2018.