Effect of coculturing canine notochordal,nucleus pulposus and mesenchymal stromal cells for intervertebral disc regeneration

IntroductionEarly degenerative changes in the nucleus pulposus (NP) are observed after the disappearance of notochordal cells (NCs). Thus, it has been suggested that NCs play an important role in maintaining the NP and may have a regenerative potential on other cells of the NP. As the number of resident NP cells (NPCs) decreases in a degenerating disc, mesenchymal stromal (stem) cells (MSCs) may be used for cell supplementation. In this study, using cells of one species, the regenerative potential of canine NCs was assessed in long-term three-dimensional coculture with canine NPCs or MSCs.MethodsCanine NCs and canine NPCs or MSCs were cocultured in alginate beads for 28 days under hypoxic and high-osmolarity conditions. Cell viability, cell morphology and DNA content, extracellular matrix production and expression of genes related to NC markers (Brachyury, KRT18) and NP matrix production (ACAN, COL2A1, COL1A1) were assessed after 1, 15 and 28 days of culture.ResultsNCs did not completely maintain their phenotype (morphology, matrix production, gene expression) during 28 days of culture. In cocultures of NPCs and NCs, both extracellular matrix content and anabolic gene expression remained unchanged compared with monoculture groups, whereas cocultures of MSCs and NCs showed increased glycosaminoglycan/DNA. However, the deposition of these proteoglycans was observed near the NCs and not the MSCs. Brachyury expression in the MSC and NC coculture group increased in time. The latter two findings indicate a trophic effect of MSCs on NCs rather than vice versa.ConclusionsNo regenerative potential of canine NCs on canine NPCs or MSCs was observed in this study. However, significant changes in NC phenotype in long-term culture may have resulted in a suboptimal regenerative potential of these NCs. In this respect, NC-conditioned medium may be better than coculture for future studies of the regenerative potential of NCs.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0569-6) contains supplementary material, which is available to authorized users.     

Mechanical stimulation to stimulate formation of a physiological collagen architecture in tissue-engineered cartilage: a numerical study

The load-bearing capacity of today's tissue-engineered (TE) cartilage is insufficient. The arcade-like collagen network in native cartilage plays an important role in its load-bearing properties. Inducing the formation of such collagen architecture in engineered cartilage can, therefore, enhance mechanical properties of TE cartilage. Considering the well-defined relationship between tensile strains and collagen alignment in the literature, we assume that cues for inducing this orientation should come from mechanical loading. In this study, strain fields prescribed by loading conditions of unconfined compression, sliding indentation and a novel loading regime of compression-sliding indentation are numerically evaluated to assess the probability that these would trigger a physiological collagen architecture. Results suggest that sliding indentation is likely to stimulate the formation of an appropriate superficial zone with parallel fibres. Adding lateral compression may stimulate the formation of a deep zone with perpendicularly aligned fibres. These insights may be used to improve loading conditions for cartilage tissue engineering.     

Cisplatin induces production of reactive oxygen species via NADPH oxidase activation in human prostate cancer cells

This study aimed to examine the roles of reactive oxygen species (ROS) in cisplatin treatment of human prostate cancer cells; hormone-sensitive LNCaP and hormone-refractory PC3 and DU145 cells. Intracellular levels of ROS and H(2)O(2) were measured and visualized using specific fluorescent probes. NADPH oxidase (NOX) activity was detected by lucigenin chemiluminescence assay. Expression levels of NOX isoforms were determined by semi-quantitative RT-PCR. Cisplatin treatment increased the intracellular levels of ROS and H(2)O(2) in three prostate cancer cell lines. The increase was transient and robust in hormone-sensitive LNCaP cells compared with hormone-refractory PC3 and DU145 cells. Consistent with these findings, the NOX activity induced by cisplatin was higher in LNCaP cells than in PC3 and DU145 cells. Expression pattern of NOX isoforms varied among three cell lines and the NOX activity was independent of NOX expression. Taken together, we have shown that cisplatin induces production of ROS and H(2)O(2) via NOX activation in human prostate cancer cell lines, which is most prominent in hormone-sensitive LNCaP cells.     

Isolation and characterization of some novel genes of the apolipoprotein A-I family in Japanese eel, <Emphasis Type="Italic">Anguilla japonica</Emphasis>

Apolipoproteins such as apolipoprotein (apo) A-I, apoA-IV, and apoE are lipid binding proteins synthesized mainly in the liver and the intestine and play an important role in the transfer of exogenous or endogenous lipids through the circulatory system. To investigate the mechanism of lipid transport in fish, we have isolated some novel genes of the apoA-I family, apoIA-I (apoA-I isoform) 1–11, from Japanese eel by PCR amplification. Some of the isolated genes of apoIA-I corresponded to 28kDa-1 cDNAs which had already been deposited into the database and encoded an apolipoprotein with molecular weight of 28 kDa in the LDL, whereas others seemed to be novel genes. The structural organization of all apoIA-Is consisted of four exons separated by three introns. ApoIA-I10 had a total length of 3232 bp, whereas other genes except for apoIA-I9 ranged from 1280 to 1441 bp. The sequences of apoIA-Is at the exon-intron junctions were mostly consistent with the consensus sequence (GT/AG) at exon-intron boundaries, whereas the sequences of 3′ splice acceptor in intron 1 of apoIA-I1-7 were (AC) but not (AG). The deduced amino acid sequences of all apoIA-Is contained a putative signal peptide and a propeptide of 17 and 5 amino acid residues, respectively. The mature proteins of apoIA-I1-3, 7, and 8 consisted of 237 amino acids, whereas those of apoIA-I4-6 consisted of 239 amino acids. The mature apoIA-I10 sequence showed 65% identity to amino acid sequence of apoIA-I11 which was associated with an apolipoprotein with molecular weight of 23 kDa in the VLDL. All these mature apoIA-I sequences satisfied the common structural features depicted for the exchangeable apolipoproteins such as apoA-I, apoA-IV, and apoE but apoIA-I11 lacked internal repeats 7, 8, and 9 when compared with other members of apoA-I family. Phylogenetic analysis showed that these novel apoIA-Is isolated from Japanese eel were much closer to apoA-I than apoA-IV and apoE, suggesting new members of the apoA-I family.     

Effect of color temperature of light sources on slow-wave sleep

In order to examine whether the spectral compositions of light source may affect sleep quality, sleep architecture under different color temperatures of light sources was evaluated. Seven healthy males were exposed to the light sources of different color temperatures (3000 K, 5000 K and 6700 K) for 6.5 h before sleep. The horizontal illuminance level was kept at 1000 lux. Subjects slept on a bed in near darkness (< 10 lux) after extinguishing the light, and polysomnograms recorded the sleep parameters. In the early phase of the sleep period, the amount of stage-4 sleep (S4-sleep) was significantly attenuated under the higher color temperature of 6700 K compared with the lower color temperature of 3000 K. Present findings suggest that light sources with higher color temperatures may affect sleep quality in a view that S4-sleep period is important for sleep quality.     

Techniques of SNP genotyping and remote sensing applied for elucidation of the contribution of polygene and environmental factors to inter-individual variation in skin pigmentation phenotype

We present a conceptual framework for applying techniques of SNP genotyping as a molecular biological approach and remote sensing as an ecological approach to elucidation of the contribution of polygene and environmental factors to inter-individual variation in skin pigmentation phenotype. Additionally, we discuss the obstacles that frustrate our efforts to identify how the human genome encodes the complex phenotype and suggest the use of computational methods designed for knowledge discovery within hereditary database.     

Analysis of expressed sequence tags in prothallia of Adiantum capillus-veneris

The analysis of expressed sequences from a diverse set of plant species has fueled the increase in understanding of the complex molecular mechanisms underlying plant growth regulation. While representative data sets can be found for the major branches of plant evolution, fern species data are lacking. To further the availability of genetic information in pteridophytes, a normalized cDNA library of Adiantum capillus-veneris was constructed from prothallia grown under white light. A total of 10,420 expressed sequence tags (ESTs) were obtained and clustering of these sequences resulted in 7,100 nonredundant clusters. Of these, 1,608 EST clusters were found to be similar to sequences of known function and 1,092 EST clusters showed similarity to sequences of unknown function. Given the usefulness of Adiantum for developmental studies, the sequence data represented in this report stand to make a significant contribution to the understanding of plant growth regulation, particularly for pteridophytes.     

Human neuronal nitric oxide synthase can catalyze one-electron reduction of adriamycin: role of flavin domain

We have analyzed the mechanism of one-electron reduction of adriamycin (Adr) using recombinant full-length human neuronal nitric-oxide synthase and its flavin domains. Both enzymes catalyzed aerobic NADPH oxidation in the presence of Adr. Calcium/calmodulin (Ca(2+)/CaM) stimulated the NADPH oxidation of Adr. In the presence or absence of Ca(2+)/CaM, the flavin semiquinone radical species were major intermediates observed during the oxidation of the reduced enzyme by Adr. The FAD-NADPH binding domain did not significantly catalyze the reduction of Adr. Neither the FAD semiquinone (FADH*) nor the air-stable semiquinone (FAD-FMNH*) reacted rapidly with Adr. These data indicate that the fully reduced species of FMN (FMNH(2)) donates one electron to Adr, and that the rate of Adr reduction is stimulated by a rapid electron exchange between the two flavins in the presence of Ca(2+)/CaM. Based on these findings, we propose a role for the FAD-FMN pair in the one-electron reduction of Adr.