Osteogenic differentiation of oBMSCs.

<p>(A) Ovine bone marrow mesenchymal stromal cells (oBMSCs, n = 4, passages 2^nd-5^th) at day 0 of differentiation (left) and after culture for 21 days in non-differentiation control medium (20% FBS/DMEM, middle) and osteogenic-differentiation medium (right) (Magnification x100). The presence of osteoblasts was assessed by detection of calcium deposits using Alizarin Red (AR) stain (Magnification x200). (B) Bar graph represents the percentage of area positively stained for AR expressed as the mean ± standard error. 1. * and # indicates p<0.05. (C) The osteogenic differentiation potential was confirmed by quantitative Real Time Polymerase Chain Reaction (qRT-PCR). The expressions of osteogenic- and multipotency-specific genes were analyzed. mRNA levels were measured in three independent samples (n = 3) by qRT-PCR as described in Materials and Methods. Data are expressed as mean ± standard error of the relative expression (REL). The results are normalized to values obtained for oBMSCs at day 0 of differentiation, considered to equal 1.</p

Primers used for the relative expression (REL) of typical genes for differentiation and multipotency.

<p>Primers used for the relative expression (REL) of typical genes for differentiation and multipotency.</p

Adipogenic differentiation of oBMSCs.

<p>(A) Ovine bone marrow mesenchymal stromal cells (oBMSCs, n = 4, passages 2^nd-5^th) at day 0 (left) of differentiation and cultured for 21 days in non-differentiation control medium (20% FBS/DMEM; middle) and adipogenic-differentiation medium (right). The presence of adipocytes was assessed by detection of lipid drops using Oil Red O (OR-O) stain. Magnification x200. (B) Bar graph represents the percentage of area positively stained for OR-O expressed as the mean ± standard error. * and ** indicates p<0.05. (C). mRNA levels were measured in three independent samples (n = 3) by quantitative Real Time Polymerase Chain Reaction (qRT-PCR), as described in Materials and Methods. Data are expressed as mean ± standard error of the relative expression (REL). The results are normalized to values obtained for oBMSCs at day 0 of differentiation, considered to equal 1.</p

Osteogenic differentiation of ovine bone marrow mesenchymal stromal cells (oBMSCs) cultured on Col I sponges and β-tricalcium phosphate (bTCP) ceramic.

<p>(A) Histological evaluation of osteogenic constructs (oBMSCs at passages 3^rd and 4^th, n = 3) stained with hematoxylin-eosin (HE), Alizarin Red (AR) and Von Kossa (VK). Magnification x100. (B) Immunohistochemical analysis of the osteogenic constructs immunostained for type I collagen (Col I) and osteocalcin (OCN). Magnification x100. (C) Bar graph represents the percentage of cells positive for AR, VK staining and OCN and Col I immunostaining, expressed as the mean ± standard error. * indicates p<0.05</p

Energy dispersive X-ray (EDX) analysis performed on the type I collagen (Col I) constructs.

<p>(A) Images obtained by scanning electron microscopy (SEM) showing the two areas analyzed; the number “1” indicates a precipitate, and the number “2” indicates a cell aggregate. (B) Spectrum of the two regions analyzed showing the elements detected: Spectrum 1 corresponding with a precipitate and spectrum 2 with a cell aggregate. (C) Bar graph represents the percentage of the elements found in five different analyzed spectra; three from the analyzed precipitate and the other two from the cell aggregate. (D) Map of C, Ca and P distribution analyzed in the whole sample.</p

Chondrogenic differentiation of oBMSCs.

<p>(A) Ovine bone marrow mesenchymal stromal cells (oBMSCs, n = 3, passages 2^nd-5^th) in micromass culture for 21 days in non-differentiation control medium (“t21 Control”; 20% FBS/DMEM) and chondrogenic-differentiation medium (“t21 Chondro”) (Magnification x100 and x200, respectively). Micromasses were stained with hematoxylin-eosin (HE), Masson´s trichrome (MT), PAS-Alcian blue (PAS-AB) and safranin O (SO). Immunodetection of type I and type II collagen (Col I and Col II) was assessed on micromasses. (B) The chondrogenic differentiation potential was confirmed by quantitative Real Time Polymerase Chain Reaction (qRT-PCR). The expressions of chondrogenic- and multipotency-specific genes were analyzed. Data are expressed as mean ± standard error of the relative expression (REL). The results are normalized to values obtained for oBMSCs at day 0 of differentiation, considered to equal 1.</p

Morphologic and phenotypic characterization of oBMSCs.

<p>(A) Image of ovine bone marrow mesenchymal stromal cells at passage 5 and 8 isolated from iliac crest aspirates. (B) Image of human bone marrow mesenchymal stromal cells (hBMSCs) at passage 10 isolated from bone marrow aspirates, as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171231#pone.0171231.ref033" target="_blank">33</a>]. Both images show the typical spindle-shaped fibroblast-like morphology characteristic of the MSC. Original magnification x100. (C) Phenotypic characterization by flow cytometry of a representative population of oBMSCs, for markers characteristic of MSCs and hematopoietic cells that react with sheep. The purple line signifies the specific antibody, while the orange line represents the isotype control. Plots correspond to a representative experiment (D) Bar graph representing the percentage average values (mean ± standard error) of positivity of the markers analyzed from 3 samples at different passages (at 3^rd, 4^th and 8^th passages); markers characteristic of MSCs (CD29, CD44, CD166), embryonic cells (SSEA-4), and hematopoietic cells (CD45).</p

Osteogenic differentiation of oBMSCs.

<p>(A) Ovine bone marrow mesenchymal stromal cells (oBMSCs, n = 4, passages 2^nd-5^th) at day 0 of differentiation (left) and after culture for 21 days in non-differentiation control medium (20% FBS/DMEM, middle) and osteogenic-differentiation medium (right) (Magnification x100). The presence of osteoblasts was assessed by detection of calcium deposits using Alizarin Red (AR) stain (Magnification x200). (B) Bar graph represents the percentage of area positively stained for AR expressed as the mean ± standard error. 1. * and # indicates p<0.05. (C) The osteogenic differentiation potential was confirmed by quantitative Real Time Polymerase Chain Reaction (qRT-PCR). The expressions of osteogenic- and multipotency-specific genes were analyzed. mRNA levels were measured in three independent samples (n = 3) by qRT-PCR as described in Materials and Methods. Data are expressed as mean ± standard error of the relative expression (REL). The results are normalized to values obtained for oBMSCs at day 0 of differentiation, considered to equal 1.</p

Scanning electron microscopy (SEM).

<p>SEM images obtained from type I collagen (Col I) and β-tricalcium phosphate (bTCP) ceramic constructs (oBMSCs at passages 3^rd and 4^th, n = 2). Cell-free Col I sponges (W/O CELLS, A-E) and Col I constructs with cells (W/CELLS, F-J) are shown in the two first rows. Cell-free bTCP scaffolds (W/O CELLS, K-O) and bTCP constructs with cells (W/ CELLS, P-T) are shown in the last two rows. Scales of bars: K, P 700 μm; L, Q 400 μm; A, F, M, R 200 μm; B, G, N, S 50 μm; C, H 40 μm; D, I 20 μm; E, J, O, T 10 μm. Black arrows: cells; black stars: extracellular matrix; red stars: cell prolongations.</p