Selection of reference genes for normalization of quantitative real-time PCR in organ culture of the rat and rabbit intervertebral disc

<p>Abstract</p> <p>Background</p> <p>The accuracy of quantitative real-time RT-PCR (qRT-PCR) is often influenced by experimental artifacts, resulting in erroneous expression profiles of target genes. The practice of employing normalization using a reference gene significantly improves reliability and its applicability to molecular biology. However, selection of an ideal reference gene(s) is of critical importance to discern meaningful results. The aim of this study was to evaluate the stability of seven potential reference genes (Actb, GAPDH, 18S rRNA, CycA, Hprt1, Ywhaz, and Pgk1) and identify most stable gene(s) for application in tissue culture research using the rat and rabbit intervertebral disc (IVD).</p> <p>Findings</p> <p><it>In vitro</it>, four genes (Hprt1, CycA, GAPDH, and 18S rRNA) in rat IVD tissue and five genes (CycA, Hprt1, Actb, Pgk1, and Ywhaz) in rabbit IVD tissue were determined as most stable for up to 14 days in culture. Pair-wise variation analysis indicated that combination of Hprt1 and CycA in rat and the combination of Hprt1, CycA, and Actb in rabbit may most stable reference gene candidates for IVD tissue culture.</p> <p>Conclusions</p> <p>Our results indicate that Hprt1 and CycA are the most stable reference gene candidates for rat and rabbit IVD culture studies. In rabbit IVD, Actb could be an additional gene employed in conjunction with Hprt1 and CycA. Selection of optimal reference gene candidate(s) should be a pertinent exercise before employment of PCR outcome measures for biomedical research.</p

The presence of extracellular matrix degrading metalloproteinases during fetal development of the intervertebral disc

Matrix metalloproteinases (MMPs) regulate connective tissue architecture and cell migration through extracellular matrix (ECM) degradation and are associated with both physiological and pathological processes. Although they are known to play a role in skeletal development, little is known about the role of MMPs in intervertebral disc (IVD) development. Sixteen fetal human lumbar spine segments, obtained at autopsy, were compared with five normal, non-fetal L4–L5 IVDs. Intensity and/or localization of immunohistochemical staining for MMP-1, -2, -3 and -14 were evaluated by three independent observers. MMP-2 production and activation was quantified by gelatin zymography. MMP-1 and -14 were abundantly present in the nucleus pulposus (NP) and notochordal (NC) cells of the fetal IVDs. In non-fetal IVDs, MMP-1 and -14 staining was significantly less intense (p = 0.001 and p < 0.001, respectively). MMP-3 was found in almost the entire IVD with no significant difference from non-fetal IVDs. MMP-2 staining in the NC and NP cells of the fetal IVD was moderate, but weak in the non-fetal IVD. Gelatin zymography showed a negative correlation of age with MMP-2 activity (p < 0.001). MMP-14 immunostaining correlated positively with MMP-2 activity (p = 0.001). For the first time, the presence of MMP-1, -2, -3 and -14 in the fetal human IVD is shown and the high levels of MMP-1, -2 and -14 suggest a role in the development of the IVD. In particular, the gradual decrease in MMP-2 activation during gestation pinpoints this enzyme as key player in fetal development, possibly through activation by MMP-1 and -14

An understanding of intervertebral disc development, maturation and cell phenotype provides clues to direct cell-based tissue regeneration therapies for disc degeneration

Cell-based regenerative medicine therapies have been proposed for repairing the degenerated intervertebral disc (a major cause of back pain). However, for this approach to be successful, it is essential to characterise the phenotype of its native cells to guarantee that implanted cells differentiate and maintain the correct phenotype to ensure appropriate cell and tissue function. While recent studies have increased our knowledge of the human nucleus pulposus (NP) cell phenotype, their ontogeny is still unclear. The expression of notochordal markers by a subpopulation of adult NP cells suggests that, contrary to previous reports, notochord-derived cells are retained in the adult NP, possibly coexisting with a second population of cells originating from the annulus fibrosus or endplate. It is not known, however, how these two cell populations interact and their specific role(s) in disc homeostasis and disease. In particular, notochordal cells are proposed to display both anabolic and protective roles; therefore, they may be the ideal cells to repair the degenerate disc. Thus, understanding the ontogeny of the adult NP cells is paramount, as it will inform the medical and scientific communities as to the ideal phenotype to implant into the degenerate disc and the specific pathways involved in stem cell differentiation towards such a phenotype. © 2014 The Author(s)

Trockenbearbeitung mit Cermetwerkzeugen im Schwerpunkt Oberflaechen- und Schichttechnologie. Teilprojekt A: Substratentwicklung und Bearbeitung von Bohrern Abschlussbericht

Available from TIB Hannover: F97B1690+a / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEBundesministerium fuer Bildung, Wissenschaft, Forschung und Technologie, Bonn (Germany)DEGerman

Noninvasive 3D vital imaging and characterization of notochordal cells of the intervertebral disc by femtosecond near-infrared two-photon laser scanning microscopy and spatial-volume rendering.

The central region of the intervertebral disc (IVD) in infant humans is made and maintained by notochordal cells (NCs). These cells disappear during maturation to be replaced by mature chondrocyte-like cells. NCs are completely different morphologically from the mature chondrocyte-like IVD cells and have complex and essential functions but little is known about them. Recently, two-photon laser scanning microscopy (TPLSM) using near-infrared (NIR) femtosecond pulsed lasers has emerged as a promising noninvasive optical technique for observing unfixed living 3D biological specimens in situ and in vitro. Several lines of evidence suggest that compared with conventional laser scanning confocal microscopy (LSCM), femtosecond NIR laser-based TPLSM has any number of advantages including 3D resolution without a spatial filter (confocal pinhole), minimal photobleaching, and photodamage above and below the focal plane, and importantly, greater depth penetration. We have thus taken advantage of these unique features of femtosecond laser-based TPLSM for vital 3D imaging in conjunction with advanced spatial-volume rendering modalities to compare morphologies of NCs/clusters from pig caudal discs with chondrocyte-like IVD cells from bovine caudal discs, both in ex vivo tissue and when isolated and grown in vitro within 3D alginate scaffolds. Our results provide evidence that (a) ex vivo notochordal tissue consists of areas with NC clusters, and those dominated by tubular structures of low cell density (b) within 3D in vitro scaffolds the morphology of NC is heterogeneous and the cells contain distinct cytoplasmic vacuole-like structures occasionally including acidic subinclusions (c) a quantitative determination based on 3D spatial and volumetric-rendering reveals an average NC diameter of 22.05 microm (range 11.96-46.63 microm) and NC volume of 9701 microm(3) (2041-36427 microm(3)) whereas chondrocyte-like cells have a mean volume of 3279 microm(3) and diameter of 12.20 microm. Taken together, this study demonstrates that femtosecond TPLSM has unique advantages over other conventional histological and in particular LSCM for high resolution noninvasive vital characterization of notochordal and chondrocyte-like cells of IVD over extended depths beyond 300-500 microm

Diamantbeschichtung komplexer Geometrien Funktionelle Eigenschaften von Diamantschichten auf Zerspanungswerkzeugen

Available from TIB Hannover: DtF QN1(27,26) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEBundesministerium fuer Forschung und Technologie (BMFT), Bonn (Germany)DEGerman