The performance of saliva test strips for determining ethanol levels, as compared to gas chromatography and breathalyser methods.

We set out to determine the performance of the Testi Technologies enzymatic assay saliva ethanol test strips of three different detection levels: 0 g/L, 0.2 g/L and 0.5 g/L, using as the reference method a gas chromatography analyser (GC). Alcohol levels were measured in 104 volunteers at up to three points in time, using up to three test strips per measurement, while gathering blood samples and breathalyser readings in parallel. The plasma alcohol concentrations (PAC) were determined from the plasma samples using GC. The qualitative results of the test strips were compared to the quantitative results from the reference method, as well as the breathalyser readings, and the amount of true and false positive and true and false negative results were classified using predetermined cut-off levels. The best performing test strips were the 0 g/L and the 0.2 g/L strips. The 0 g/L strips had a sensitivity and specificity of 1.00, as false negatives and false positives were not detected. The 0.2 g/L strips had a sensitivity and specificity [95% confidence interval (CI)] of 0.98 (0.96 - 1.00) and 0.83 (0.62 - 1.00) respectively, an accuracy of 0.97 (0.95 - 0.99), and a diagnostic odds ratio of 205.00 (35.33 - 1189.66). The test strips perform their intended purpose of screening for alcohol consumption well, with their great sensitivity as a defining property compared to other testing methods. For them to be able to be implemented in a clinical setting however, further refinement of the tests' characteristics would be required.

Challenges in Fabrication of Tissue-Engineered Cartilage with Correct Cellular Colonization and Extracellular Matrix Assembly.

A correct articular cartilage ultrastructure regarding its structural components and cellularity is important for appropriate performance of tissue-engineered articular cartilage. Various scaffold-based, as well as scaffold-free, culture models have been under development to manufacture functional cartilage tissue. Even decellularized tissues have been considered as a potential choice for cellular seeding and tissue fabrication. Pore size, interconnectivity, and functionalization of the scaffold architecture can be varied. Increased mechanical function requires a dense scaffold, which also easily restricts cellular access within the scaffold at seeding. High pore size enhances nutrient transport, while small pore size improves cellular interactions and scaffold resorption. In scaffold-free cultures, the cells assemble the tissue completely by themselves; in optimized cultures, they should be able to fabricate native-like tissue. Decellularized cartilage has a native ultrastructure, although it is a challenge to obtain proper cellular colonization during cell seeding. Bioprinting can, in principle, provide the tissue with correct cellularity and extracellular matrix content, although it is still an open question as to how the correct molecular interaction and structure of extracellular matrix could be achieved. These are challenges facing the ongoing efforts to manufacture optimal articular cartilage.

Effects of long-term low oxygen tension in human chondrosarcoma cells.

The cell-based therapies could be potential methods to treat damaged cartilage tissues. Instead of native hyaline cartilage, the current therapies generate mainly weaker fibrocartilage-type of repair tissue. A correct microenvironment influences the cellular phenotype, and together with external factors it can be used, for example, to aid the differentiation of mesenchymal stem cells to defined types of differentiated adult cells. In this study, we investigated the effect of long-term exposure to 5% low oxygen atmosphere on human chondrosarcoma HCS-2/8 cells. This atmosphere is close to normal oxygen tension of cartilage tissue. The proteome was analyzed with label-free mass spectrometric method and further bioinformatic analysis. The qRT-PCR method was used to gene expression analysis, and ELISA and dimethylmethylene blue assays for type II collagen and sulfated glycosaminoglycan measurements. The 5% oxygen atmosphere did not influence cell proliferation, but enhanced slightly ACAN and COL2A1 gene expression. Proteomic screening revealed a number of low oxygen-induced protein level responses. Increased ones included NDUFA4L2, P4HA1, NDRG1, MIF, LDHA, PYGL, while TXNRD1, BAG2, TXN2, AQSTM1, TNFRSF1B, and EPHX1 decreased during the long-term low oxygen atmosphere. Also a number of proteins previously not related to low oxygen tension changed during the treatment. Of those S100P, RPSS26, NDUFB11, CDV3, and TUBB8 had elevated levels, while ALCAM, HLA-B, EIF1, and ACOT9 were lower in the samples cultured at low oxygen tension. In conclusion, low oxygen condition causes changes in the cellular amounts of several proteins.

Rho-kinase inhibitor Y-27632 and hypoxia synergistically enhance chondrocytic phenotype and modify S100 protein profiles in human chondrosarcoma cells.

Articular chondrocytes are slowly dividing cells that tend to lose their cell type-specific phenotype and ability to produce structurally and functionally correct cartilage tissue when cultured. Thus, culture conditions, which enhance the maintenance of chondrocyte phenotype would be very useful for cartilage research. Here we show that Rho-kinase inhibition by Y-27632 under hypoxic conditions efficiently maintains and even enhances chondrocyte-specific extracellular matrix production by chondrocytic cells. The effects of long-term Y-27632 exposure to human chondrosarcoma 2/8 cell phenotype maintenance and extracellular matrix production were studied at normoxia and at a 5% low oxygen atmosphere. Y-27632 treatment at normoxia induced ACAN and COL2A1 gene up-regulation and a minor increase of sulfated glycosaminoglycans (sGAGs), while type II collagen expression was not significantly up-regulated. A further increase in expression of ACAN and COL2A1 was achieved with Y-27632 treatment and hypoxia. The production of sGAGs increased by 65.8%, and ELISA analysis revealed a 6-fold up-regulation of type II collagen. Y-27632 also induced the up-regulation of S100-A1 and S100-B proteins and modified the expression of several other S100 protein family members, such as S100-A4, S100-A6, S100-A13 and S100-A16. The up-regulation of S100-A1 and S100-B proteins is suggested to enhance the chondrocytic phenotype of these cells.

Rho-kinase inhibitor Y-27632 increases cellular proliferation and migration in human foreskin fibroblast cells.

The idea of direct differentiation of somatic cells into other differentiated cell types has attracted a great interest recently. Rho-kinase inhibitor Y-27632 (ROCKi) is a potential drug molecule, which has been reported to support the gene expressions typical for the chondrocytes, thus restricting their phenotypic conversion to fibroblastic cells upon the cellular expansion. In this study, we have investigated the short-term biological responses of ROCKi to human primary foreskin fibroblasts. The fibroblast cells were exposed to 1 and 10 µM ROCKi treatments. A proteomics analysis revealed expression changes of 56 proteins, and a further protein pathway analysis suggested their association with the cell morphology, the organization, and the increased cellular movement and the proliferation. These functional responses were confirmed by a Cell-IQ time-lapse imaging analysis. Rho-kinase inhibitor treatment increased the cellular proliferation up to twofold during the first 12 h, and a wound model based migration assay showed 50% faster filling of the mechanically generated wound area. Additionally, significantly less vinculin-associated focal adhesions were present in the ROCKi-treated cells. Despite the marked changes in the cell behavior, ROCKi was not able to induce the expression of the chondrocyte-specific genes, such as procollagen α1 (II) and aggrecan.

Proteomics of chondrocytes with special reference to phosphorylation changes of proteins in stretched human chondrosarcoma cells.

For proteomic analysis, cartilage molecular composition is a challenging mixture of highly glycosylated proteoglycans and triple-helical collagens, which constitute the major part of cartilage macromolecules. Selective separation of these molecules from the minor components is generally needed before mass spectrometry-based identification of lower-abundancy proteins is possible. The cell density of cartilage is also very low, therefore, cell cultures offer an easier approach to study cellular responses of chondrocytic cells, e.g., to mechanical stimuli. In this study, we investigated the phosphorylation events in human chondrosarcoma cells during cellular stretching. Human chondrosarcoma cells were stretched to 8% strain at a frequency of 1 Hz. One set of experiments included cellular stretching which lasted 2 hours, and the other one included experiments of 2 hours daily treatment for up to 3 days. Two-dimensional polyacrylamide gel electrophoresis combined with chromatographic phosphoprotein pre-enrichment and electrospray ionization mass spectrometry-based protein identification was used to reveal changes of phosphoproteins in cells exposed to cyclic stretching. We discovered that 2 hours cyclic stretching increased the phosphorylation of moesin, elongation factor eEF1D and leprecan, while the phosphorylation of elongation factor eEF1B decreased after cellular stretching. Western blot analyses with phospho-specific antibodies suggested that stretching induces phosphorylation of ERK of the MAP kinase pathway, but did not induce phosphorylation of phosphatidylinositol 3-kinase. In conclusion, the proteomic approach revealed that cellular stretching induced specific phosphorylation changes in chondrosarcoma cells.