Images showing the results obtained when cells were cultured on the smooth surface.

<p>(<b>A</b>) smooth surface at SEM (Calibration Bar = 10 µm); (<b>B</b>) cells, cultured on a smooth surface, under SEM, show an elongated shape (Calibration Bar = 10 µm); (<b>C</b>) confocal image showing the relationship between cells and the smooth surface (Calibration Bar = 10 µm); (<b>D</b>) image showing the relationship between cells and the different surface texturing: (1) concave and (2) smooth.</p

Immunofluorescence for HLA-1 Human FITC (green) (A) <i>In vivo</i> concave PLGA scaffold.

<p>(<b>B</b>) <i>In vivo</i> smooth PLGA scaffold. (Calibration Bars = 5 µm). Nuclear staining is obtained with DAPI (blue).</p

Images showing primary and secondary micro concavities at scanning electron and confocal microscopy.

<p>(<b>A</b>) Primary micro concavity (<u>arrow</u>) of the PLGA surface at SEM. Cells can be completely contained within a primary concavity, due to its dimensions. (Calibration Bar = 10 µm); (<b>B</b>) SEM analysis of primary concavity dimensions (Calibration Bar = 10 µm); (<b>C</b>) SEM analysis of secondary concavity dimensions (Calibration Bar = 10 µm); (<b>D</b>) The interaction between the concave surface, showing primary (<u>white arrow</u>) and secondary (<u>red arrows</u>) micro-concavities at the confocal microscope (in green a cell within a concavity). The intimate adherence of a cell to the polymer surface and its nuclear polarity are clearly observable. The image was been obtained superimposing dark field with light field confocal microscopy (Calibration Bar = 10 µm); (<b>E</b>) Confocal image showing primary (outlined in red) and secondary (outlined in blue) micro-concavities and spider-shaped cellular elongations (Calibration Bar = 10 µm); (<b>F</b>) A gingival fibroblast not showing cellular alterations or nuclear polarity at the confocal microscope (Calibration Bar = 10 µm).</p

ELISA assays performed on (A) VEGF and (B) BMP2 released within the supernatant by SBP-DPSCs after 24, 48, 72 and 96h from plating on smooth and concave textured surface; ELISA assays performed on (C) VEGF and (D) BMP2 present in the cell layer of SBP-DPSCs after 24, 48, 72 and 96h from plating on smooth and concave texturing.

<p>Each experiment was performed in triplicate (n = 3). The error bars are ±SD. *p<0.01. N indicates samples not NMP-treated; P indicates NMP-treated samples.</p

Haematoxilin and Eosin staining.

<p>(<b>A</b>) <i>In vivo</i> sample on concave PLGA scaffold (Calibration Bar = 10 µm). The figures shows that the bone tissue is of great thickness with an evident periosteal layer (red arrow) and containing osteocytes entrapped within the matrix (black arrows). (<b>B</b>) <i>In vivo</i> sample on smooth PLGA scaffold (Calibration Bar = 5 µm) appeared to be more primitive, thin (black arrow) and not as well developed above a connective loose tissue containing vessels (red arrow).</p

Alkaline phosphatase detection during cell differentiation.

<p>The image shows the quantity of ALP during osteoblast differentiation at 24, 48, 72 and 96 hours within the cells cultured on the different surfaces. The data have been rounded to the closest integer value. The error bars are ±SD.*p<0.01. Each experiment was performed in triplicate (n = 3).</p

Immunofluorescence confirming the presence of a mineralized extra cellular matrix on concave texturing.

<p>The panel shows positivity for Collagen I (<b>A</b>) FITC (green) (Calibration Bar = 10 µm), BAP [Bone Alkaline Phosphatase] (<b>B</b>) FITC (green) (Calibration Bar = 5 µm), OC [Osteocalcin] (<b>C</b>) PE (red) (Calibration Bar = 7 µm), ON [Osteonectin] (<b>D</b>) FITC(green) (Calibration Bar = 7 µm) and BSP [Bone Sialoprotein] (<b>E</b>) PE (red) (Calibration Bar = 3 µm). The same analysis confirming the presence of a mineralized extra cellular matrix on smooth texturing. The panel shows positivity for Collagen I (<b>F</b>) FITC (green) (Calibration Bar = 5 µm), BAP (<b>G</b>) FITC (green) (Calibration Bar = 7 µm), OC (<b>H</b>) PE (red) (Calibration Bar = 3 µm), ON (<b>I</b>) FITC (green) (Calibration Bar = 7 µm) and BSP (<b>J</b>) PE (red) (Calibration Bar = 3 µm). Nuclear staining is obtained with DAPI (blue).</p

Unsuspected osteochondroma-like outgrowths in the cranial base of Hereditary Multiple Exostoses patients and modeling and treatment with a BMP antagonist in mice

<div><p>Hereditary Multiple Exostoses (HME) is a rare pediatric disorder caused by loss-of-function mutations in the genes encoding the heparan sulfate (HS)-synthesizing enzymes EXT1 or EXT2. HME is characterized by formation of cartilaginous outgrowths—called osteochondromas- next to the growth plates of many axial and appendicular skeletal elements. Surprisingly, it is not known whether such tumors also form in endochondral elements of the craniofacial skeleton. Here, we carried out a retrospective analysis of cervical spine MRI and CT scans from 50 consecutive HME patients that included cranial skeletal images. Interestingly, nearly half of the patients displayed moderate defects or osteochondroma-like outgrowths in the cranial base and specifically in the clivus. In good correlation, osteochondromas developed in the cranial base of mutant <i>Ext1</i>^<i>f/f</i><i>;Col2-CreER</i> or <i>Ext1</i>^<i>f/f</i><i>;Aggrecan-CreER</i> mouse models of HME along the synchondrosis growth plates. Osteochondroma formation was preceded by phenotypic alteration of cells at the chondro-perichondrial boundary and was accompanied by ectopic expression of major cartilage matrix genes -<i>collagen 2</i> and <i>collagen X-</i> within the growing ectopic masses. Because chondrogenesis requires bone morphogenetic protein (BMP) signaling, we asked whether osteochondroma formation could be blocked by a BMP signaling antagonist. Systemic administration with LDN-193189 effectively inhibited osteochondroma growth in conditional <i>Ext1</i>-mutant mice. In vitro studies with mouse embryo chondrogenic cells clarified the mechanisms of LDN-193189 action that turned out to include decreases in canonical BMP signaling pSMAD1/5/8 effectors but interestingly, concurrent increases in such anti-chondrogenic mechanisms as pERK1/2 and <i>Chordin</i>, <i>Fgf9</i> and <i>Fgf18</i> expression. Our study is the first to reveal that the cranial base can be affected in patients with HME and that osteochondroma formation is amenable to therapeutic drug intervention.</p></div

Osteochondroma development in long bones and ribs in juvenile mice is inhibited by systemic LDN-193189 treatment.

<p><b>(A to D)</b> μCT images (A, B and D) and X-ray image (C) of knee and ribs from mutant <i>Ext1</i>^<i>f/f</i><i>;Agr-CreER</i> mice receiving vehicle treatment and showing large osteochondromas (double arrowheads). (<b>E to H</b>) Analogous images from companion mutant mice receiving LDN treatment for 6 weeks. Note the appreciable reduction in osteochondorma size in both knee and ribs (arrowhead). Bar in (A) for A, B, E and F, 5 mm; bar in (C) for C and G, 3 mm; and bar in (D) for D and H, 1.5 mm.</p

Stereotypic osteochondromas form in a juvenile HME mouse model.

<p>(<b>A-F</b>) Bright field and fluorescence images of longitudinal sections of spheno-occipital (<i>sos</i>) synchondroses (A-C), tibia (D-E) and ribs (F) from 5 week-old control <i>R26-tdTomato;Agr-CreER</i> mice that had been injected with vehicle 3 days earlier. The fluorescence images (B and E) show absence of reporter activity in growth plates (<i>gp</i>) and perichondrium (<i>pc</i>) at any anatomical site examined and thus, lack of <i>CreER</i> leakage in this model. Note that ribs contain a large resting cartilage (<i>rc</i>) adjacent to the growth plate (F). (<b>G-H</b>) μCT images of cranial base and knee from control <i>Ext1</i>^<i>f/f</i><i>;Agr-CreER</i> mice sacrificed 6 weeks from vehicle injection and displaying normal and typical anatomical characteristics. (<b>I-N</b>) Bright field and fluorescence images from companion <i>R26-tdTomato;Agr-CreER</i> mice that were administered tamoxifen once and were sacrificed 3 days post-injection. Note that reporter activity is very strong in spheno-occipital synchondrosis (<i>sos</i>), tibia and rib growth plates and rib resting cartilage (J, K, M and N) and is also conspicuous in flanking perichondrial (<i>pc</i>) cells at each anatomical location (arrowheads in K and M). Boxed area in J is shown at higher magnification in K. (<b>O-P</b>) μCT images of cranial base and knee from mutant <i>Ext1</i>^<i>f/f</i><i>;Agr-CreER</i> mice sacrificed 6 weeks after tamoxifen injection. Note the large multiple osteochondromas (arrowheads) protruding away from the bone surfaces at each anatomical site and better appreciable when contrasted to corresponding images from companion controls (G-H). Bar in (A) for A, B, I and J: 300 μm; bar in (C for C and K, 150 μm; bar in (D) for D, E, L and M, 250 μm; bar in (F) for F and N, 150 μm; and bar in (G) for G and O, 0.8 mm; and in (H) for H and P, 5 mm.</p