Additional file 1: of Human osteochondritis dissecans fragment-derived chondrocyte characteristics ex vivo, after monolayer expansion-induced de-differentiation, and after re-differentiation in alginate bead culture

mRNA expression values for collagen types I, II, and X (Col I, Col II, and Col X, respectively) and aggrecan as well as cell viability. (DOC 40 kb

Mesenchymal Stromal Cells for Sphincter Regeneration: Role of Laminin Isoforms upon Myogenic Differentiation

<div><p>Multipotent mesenchymal stromal cells (MSCs) are well known for their tri-lineage potential and ability to differentiate <i>in vitro</i> into osteogenic, chondrogenic or adipogenic lineages. By selecting appropriate conditions MSCs can also be differentiated <i>in vitro</i> into the myogenic lineage and are therefore a promising option for cell-based regeneration of muscle tissue such as an aged or damaged sphincter muscle. For the differentiation into the myogenic lineage there is still a need to evaluate the effects of extracellular matrix proteins such as laminins (LM) which are crucial for different stem cell types and for normal muscle function. The laminin family consists of 16 functionally different isoforms with LM-211 being the most abundant isoform of adult muscle tissues. In the sphincter tissue a strong expression of the isoforms LM-211/221, LM-411/421 and LM-511/521 can be detected in the different cell layers. Bone marrow-derived MSCs in culture, however, mainly express the isoforms LM-411 and LM-511, but not LM-211. Even after myogenic differentiation, LM-211 can hardly be detected. All laminin isoforms tested (LM-211, LM-411, LM-511 and LM-521) showed a significant inhibition of the proliferation of undifferentiated MSCs but, with the exception of LM-521, they had no influence on the proliferation of MSCs cultivated in myogenic medium. The strongest cellular adhesion of MSCs was to LM-511 and LM-521, whereas LM-211 was only a weakly-adhesive substrate for MSCs. Myogenic differentiation of MSCs even reduced the interaction with LM-211, but it did not affect the interaction with LM-511 and LM-521. Since during normal myogenesis the latter two isoforms are the major laminins surrounding developing myogenic progenitors, α5 chain-containing laminins are recommended for further improvements of myogenic differentiation protocols of MSCs into smooth muscle cells.</p></div

Influence of recombinant laminin isoforms on the proliferation rate of MSCs.

<p>MSCs in early passages were grown for seven days in expansion medium (GMP+<b>)</b> with or without 10 μg/ml of the recombinant laminin isoforms LM-211, LM-411, LM-511 and LM-521. All laminin isoforms decreased the proliferation rate of MSCs. In contrast, cultivation of MSCs in myogenic differentiation medium with the different recombinant laminin isoforms had no significant effect on the proliferation rate with LM-521 being the exception (n = 3 donors; experiments performed in duplicates; error bars indicate standard error of the mean; one-way ANOVA analysis; **p<0.01; ***p<0.001 in comparison to control).</p

Laminin expression by primary MSCs, the cell line HITB5 and tissue-derived HBdSMC.

<p>RT-PCR analyses (A) and immunofluorescence staining (B) of undifferentiated MSCs (Undiff), myogenically differentiated MSCs (Myo), the cell line HITB5 and HBdSMC in early passages suggested the expression of several laminin isoforms. The highest expression was observed for the α4, α5, β1 and γ1 chains. The α2 chain was only weakly expressed. Cell nuclei were counterstained in blue with DAPI (bars: 50 μm).</p

Cell adhesion to plastic-immobilized laminin isoforms.

<p>Cell-matrix interactions with undifferentiated MSCs (Undiff), myogenically differentiated MSCs (Myo), the cell line HITB5 and HBdSMC were quantitatively determined by single cell force measurement (n = 9 cells) and qualitatively by spotting assays on different recombinant laminin isoforms. LM-511 and LM-521 were the strongest adhesive substrates for smooth muscle cells and undifferentiated MSCs. Myogenic differentiation diminished the binding of MSCs to LM-411. LM-211 was only a weakly-adhesive substrate for smooth muscle cells and MSCs, and upon myogenic differentiation the adhesive capacity of LM-211 was further diminished (bar: 200 μm; error bars indicate standard error of the mean; one-way ANOVA analysis; *p<0.05; **p<0.01; ***p<0.001 in comparison to HITB5).</p

Expression of laminin chains in the mini-pig sphincter tissue.

<p>Cryostat sections of an isolated mini-pig sphincter tissue were either stained with hematoxylin-eosin (HE) or labeled with laminin chain specific antibodies (red) together with an antiserum against von Willebrand factor (vWF; green) labeling endothelial cells. By HE staining the inner epithelial lining (E), mucosa, longitudinal smooth muscle layer (LM) and circular smooth muscle layer (CM) can be distinguished. The laminin α chains (α2, α4, α5), β chains (β1, β2) and the γ1 chain are expressed in the mucosa, in the different muscular layers and around endothelial cells, albeit with different intensities. In the merged pictures the weaker expression of the laminin α2, β1 and γ1 chains in some endothelial cells is hidden behind the strong signal for von Willebrand factor staining. Cell nuclei were counterstained in blue with DAPI (bar: 500 μm).</p

Inter- and intraobserver reliability of the MTM-classification for proximal humeral fractures: A prospective study-2

Re of the anatomical neck with extension into the lesser tuberosity (LB). c) incomplete articular fractures with an medial metaphyseal wedge shaped fragment at the humeral head (MB).<p><b>Copyright information:</b></p><p>Taken from "Inter- and intraobserver reliability of the MTM-classification for proximal humeral fractures: A prospective study"</p><p>http://www.biomedcentral.com/1471-2474/9/21</p><p>BMC Musculoskeletal Disorders 2008;9():21-21.</p><p>Published online 17 Feb 2008</p><p>PMCID:PMC2275241.</p><p></p

Inter- and intraobserver reliability of the MTM-classification for proximal humeral fractures: A prospective study-3

A fracture of the lesser tuberosity. b) LBG fractures = incomplete fracture of the anatomical neck with extension into the lesser tuberosity (LB) with a fracture of the greater tuberosity. c) MBG fractures = incomplete articular fractures with an medial metaphyseal fragment at the humeral head (MB) with a fracture of the greater tuberosity. d) MBL fractures = incomplete articular fractures with an medial metaphyseal fragment at the humeral head (MB) with a fracture of the lesser tuberosity. e) MBT fractures = 4-Part B fractures appear which are MB fractures in combination with a fracture of both tuberosities (G and L = T) = MBT fractures.<p><b>Copyright information:</b></p><p>Taken from "Inter- and intraobserver reliability of the MTM-classification for proximal humeral fractures: A prospective study"</p><p>http://www.biomedcentral.com/1471-2474/9/21</p><p>BMC Musculoskeletal Disorders 2008;9():21-21.</p><p>Published online 17 Feb 2008</p><p>PMCID:PMC2275241.</p><p></p

Inter- and intraobserver reliability of the MTM-classification for proximal humeral fractures: A prospective study-4

Ic neck and fracture of the greater tuberosity. c) CL fracture = complete fracture through the anatomic neck and fracture of the lesser tuberosity. d) CT fracture = complete fracture through the anatomic neck and fracture of both tuberosities (G and L = T). e) CTM fracture = complete fracture through the anatomic neck and fracture of both tuberosities and metaphyseal fracture.<p><b>Copyright information:</b></p><p>Taken from "Inter- and intraobserver reliability of the MTM-classification for proximal humeral fractures: A prospective study"</p><p>http://www.biomedcentral.com/1471-2474/9/21</p><p>BMC Musculoskeletal Disorders 2008;9():21-21.</p><p>Published online 17 Feb 2008</p><p>PMCID:PMC2275241.</p><p></p

Inter- and intraobserver reliability of the MTM-classification for proximal humeral fractures: A prospective study-1

E. a2) Vertical M fracture = isolated metaphyseal fracture coming from the anatomic neck proceeding lateral from the lesser tuberosity to distal medial through the metaphysis. b) G fracture = isolated fracture of the greater tuberosity. c) L fracture = islolated fracture of the lesser tuberosity. d) MG fracture = metaphyseal fracture and fracture of the greater tuberosity. e) ML fracture = metaphyseal fracture and fracture of the lesser tuberosity. f) MT fracture = metaphyseal fracture and fracture of both tuberosities.<p><b>Copyright information:</b></p><p>Taken from "Inter- and intraobserver reliability of the MTM-classification for proximal humeral fractures: A prospective study"</p><p>http://www.biomedcentral.com/1471-2474/9/21</p><p>BMC Musculoskeletal Disorders 2008;9():21-21.</p><p>Published online 17 Feb 2008</p><p>PMCID:PMC2275241.</p><p></p