<i>Osterix</i>-Cre Fate Mapping Marks Bone Marrow Vascular Smooth Muscle.

<p>Cre reporter expression activated by <i>OEC</i> (<b>A–C1</b> and <b>G–L</b>) and <i>ORt</i> (<b>D–F</b> and <b>M–O</b>) mice was expressed in vascular smooth muscle cells, which encompassed arterioles running longitudinally through the center of the bone marrow. Vascular smooth muscle was identified through a combination of CD31 (<b>A–F</b>) and smooth muscle actin alpha 2 (SMA) (<b>G–O</b>) immunostaining. The marking of vascular smooth muscle was restricted to the bone marrow compartment as vascular smooth muscle cells lying just outside the cortical bone surface did not contain any Cre reporter expression (<b>J–L</b>). (<b>A, A1, D, G, J, M</b>) <i>Ai9</i> Cre reporter expression shown in red. (<b>B, B1, E</b>) CD31 immunostaining shown in green. (<b>H, K, N</b>) SMA immunostaining shown in green. (<b>C, C1, F, I, L, O</b>) Merged images.</p

Visualization of <i>OEC</i> Mediated Cre Reporter Expression Inside the Bone Marrow.

<p>(<b>A–C</b>) In addition to activating Cre reporter expression (shown in white) in cells of the osteoblast lineage, <i>OEC</i> mice resulted in the broad marking of non-hematopoietic cells within the bone marrow compartment. (<b>A’–C’</b>) Hematoxylin counterstained tissue sections corresponding to images A–C. At progressively higher levels of magnification (<b>B</b> and <b>C</b>, yellow dashed box regions), Cre reporter expression is observed in cells that contribute to the stroma throughout the bone marrow. (<b>C, C’</b>) At high magnification many Cre reporter expressing cells retain long narrow processes reminiscent of bone marrow reticular cells and line vascular sinuses. (D) Expression of the Osterix EGFPCre reporter was not detected in trabecular bone osteoblasts or bone marrow in 6 week old femurs. However, expression of Osterix was detected by immunostaining in trabecular bone osteoblasts and osteocytes (E).</p

Development of a vision sensor for monitoring of geometric parameters of weld pool in pulsed GTAW

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Tecnologia, Departamento de Engenharia Mecânica, 2016.Este trabalho apresenta o desenvolvimento de um sensor para monitoramento geométrico da poça de fusão em um processo GTAW pulsado utilizando uma câmera de alta velocidade CMOS para aquisição das imagens. A configuração da câmera é tal que o seu eixo óptico é paralelo ao eixo longitudinal da tocha e a imagem da poça de solda é transmitida por meio de um conjunto de espelhos planos. Afim de reduzir o efeito da luz causada pela emissão do arco, um filtro passa alta com corte na região do infravermelho é utilizado de tal modo que a emissão do arco no espectro visível e ultravioleta são eliminados. A configuração do sensor permite a redução do tamanho da estrutura de fixação do conjunto óptico, pois utiliza as leis da reflexão para direcionar a imagem desejada até a câmera. A estrutura de sustentação dos aparatos ópticos foi projetada com a finalidade de minimizar o efeito de vibração na câmera ocasionada pelo movimento robótico e na perda de graus de orientação da tocha no processo de soldagem robotizado. Com a configuração proposta, é possível obter imagens consistentes e de qualidade da poça de fusão permitindo extrair seus dados geométricos. Neste trabalho a principal característica geométrica desejada é a largura da poça de fusão, uma vez que é fortemente correlacionada à largura do cordão resultante. Por se tratar de um processo no qual o arco está constantemente aberto, um software para sincronização das imagens baseado na análise do brilho do arco foi proposto, mostrando eficiência na seleção das imagens com menor interferência luminosa. A integração da estrutura óptica com os softwares proporcionou imagens consistentes para análise de parâmetros que futuramente podem ser usadas nos métodos de controle.This work presents the development of a sensor for monitoring weld pool geometry in a pulsed GTAW process using a high-speed CMOS camera for image acquisition. The configuration of the camera is such that its optical axis is parallel to the longitudinal axis of the torch and the weld pool image is transmitted through a set of plane mirrors. In order to reduce the effect caused by the emission of light from the arc, a long wave pass filter with cutoff in the infrared region is used such that the emission of the arc in the UV and visible spectra are eliminated. The proposed sensor configuration enabled the reduction in size of the optical assembly structure because it uses the laws of reflection to direct the desired image to the camera. The bearing structure of the optical apparatus is designed in order to minimize the effect of camera vibration caused by robotic movement and loss of degrees orientation of the robot torch at the welding process. With the proposed configuration, it is possible to obtain consistent quality images of the weld pool, allowing extract their geometric data. At this work, the main geometrical feature desired is the weld pool width, once it is strongly correlated the resulting bead width. Because it is a process in which the arch is constantly open, one software for synchronization based on the analysis of the arc brightness was proposed showing efficiency in the selection of images with less light interference. The integration of optical structure with the proposed software provided consistent images for analysis of parameters that can be used in future control methods

<i>Osterix</i>-Cre Fate Mapping Marks Bone Marrow Perineural Cells.

<p><i>OEC</i> (<b>A-i2</b>) and <i>ORt</i> (<b>J–L</b>) activated <i>Ai9</i> Cre reporter expression (red) was observed in perineural cells that formed tubes around nerve fibers (green) located in the bone marrow compartment (A–F and J–L), channels of the cortical bone (g2, h2, i2) and adjacent to the outer periosteum (g1, h1, i1). Nerve fibers were identified by immunostaining for Neurofilament M (B, E, H, h1, h2, K, green).</p

In Vitro Differentiation of OC9+ Cells Demonstrates Their Multipotent Properties.

<p>OC9+ cells were FACS isolated from day 5 stromal cultures, replated in culture, and differentiated into osteoblasts (<b>A–F, S</b>), adipocytes (<b>G–L, T</b>), and chondrocytes (<b>M–R, U</b>). (<b>A, D, G, J, M, P</b>) Detection of <i>Ai9</i> Cre reporter expression. (<b>B, E, H, K, N, Q</b>) Imaging of cultures under DIC optics. (<b>C, F, I, L, O, R</b>) Imaging of whole mount staining of cultures. (<b>S–U</b>) Temporal assessment of differentiation by quantitative RT-PCR. (<b>A–C</b>) OC9+ cells differentiated under osteogenic conditions produced a robust mineralized matrix that could be visualized under DIC optics (<b>B</b>) and by von Kossa staining (<b>C</b>). (<b>D–F</b>) OC9+ cells grown in the absence of osteogenic conditions did not produce a mineralized matrix and did not stain by von Kossa. (<b>S</b>) Osteoblast differentiation was examined at days 0, 3, 6, and 9 of culture. All osteogenic gene markers were dramatically up-regulated during differentiation including <i>Osteocalcin</i> (<i>Bglap1</i>), <i>Bone Sialoprotein</i> (<i>IBSP</i>), <i>Dentin Matrix Protein 1</i> (<i>DMP1</i>), and <i>Osterix</i>. (<b>G–I</b>) OC9+ cells differentiated under adipogenic conditions generated cells containing large lipid vesicles that could be easily observed under DIC optics (<b>H</b>) and were stained by Oil Red-O (<b>I</b>). (<b>J–L</b>) Control cultures grown under non-adipogenic conditions did not display large lipid vesicles (<b>K</b>) or stain with Oil Red-O (<b>L</b>). (<b>T</b>) Adipogenic differentiation was examined at days 0, 3, 6, and 9 of culture. All adipogenic gene markers were dramatically up-regulated during differentiation including <i>Adiponectin</i>, <i>Perilipin</i>, <i>Fatty Acid Binding Protein 4</i> (<i>FABP4</i>), and <i>Adipsin</i>. (<b>M–O</b>) OC9+ cells differentiated under chondrogenic conditions generated areas of condensing chondrocytes that were noticeable under DIC optics (<b>N</b>) and stained strongly positive for Alcian blue (<b>O</b>). (<b>P–R</b>) Control cultures grown under non-chondrogenic conditions did not generate areas of condensing chondrocytes (<b>Q</b>) and did not stain for Alcian blue (<b>R</b>). (U) Chrondrogenic differentiation was examined at days 0, 7, and 14 of culture. All chondrogenic gene markers were dramatically up-regulated during differentiation including <i>Sox9</i>, <i>Aggrecan</i>, <i>Collagen type 2 alpha I</i> (<i>Col2a1</i>), and <i>Collagen type 10 alpha 1</i> (<i>Col10a1</i>).</p

<i>Osterix</i>-Cre Fate Mapping Marks Bone Marrow Adipocytes.

<p><i>OEC</i> (<b>A–F</b>) and <i>ORt</i> (<b>G–I</b>) activated <i>Ai9</i> Cre reporter expression was observed in bone marrow adipocytes, which appear as empty, round-shaped cells in tissue section. Cre reporter expressing bone marrow adipocytes (<b>A, C, E, G, H</b>) were identified using <i>aP2-EGFPcyan</i> reporter mice (<b>B</b>, blue fluorescent adipocytes noted by white arrows), immunostaining for Perilipin (<b>D</b>, green fluorescent adipocytes noted by white arrows), and hematoxylin counterstained tissue sections revealed round empty holes (<b>I,</b> black arrows). The marking of adipocytes was restricted to the bone marrow compartment as adipose tissue located just outside the bone cortices retained no Cre reporter expression (<b>E, F</b>).</p

Transplantation of OC9+ Cells Demonstrates Their Multipotent Properties.

<p>Stromal cells derived from <i>Osterix-Cre</i>/<i>Ai9</i> mice were cultured for 5 days, loaded into a scaffold, and transplanted into a femoral skeletal defect. (<b>A</b>) Skeletal healing of the defect was monitored by x-ray over a 6 week period. (<b>B, D</b>) Detection of OC9+ cellular progeny (red) in a femoral tissue section 6 weeks after transplantation. (<b>C, E</b>) The same tissue section shown in <b>A</b> and <b>D</b> now counterstained with hematoxylin. (<b>B</b>) A low magnification image showing OC9 cellular progeny (red) contributing to cortical and trabecular bone tissue detected by DIC imaging and hematoxylin counterstaining (purple). The yellow arrow denotes the boundary between host bone and donor bone. (<b>D, E</b>) In addition to differentiating into osteoblasts and making bone tissue, higher magnification reveals that OC9+ cells contribute to bone marrow adipocytes within the marrow and perivascular cells lining the vascular sinusoids (B = bone, F = adipocytes, and S = vascular sinusoid).</p

Constitutive <i>Osterix</i>-Cre Fate Mapping Marks a Bone Marrow Stromal Cell Population.

<p>(<b>A</b>) The intercross of <i>Osterix</i>-Cre mice with <i>Ai9</i> Cre reporter mice generates dual transgenic offspring containing <i>Osterix</i>-Cre and the <i>Ai9</i> Cre reporter (red mouse). (<b>B</b>) Cre reporter expressing cells were present in the bone marrow flush, a subset of which started to adhere to the tissue culture plate within the first 24 hours. (<b>C</b>) By day 5 of stromal culture OC9+ cells (the <i>Osterix</i>-Cre mediated Cre reporter expressing population) represents a subpopulation of the adherent cell fraction and retains a mesenchymal cell morphology. (<b>D, E</b>) FACS analysis from day 5 stromal cultures showed that OC9+ cells represent 15–20% of the total cell population. (<b>F–H</b>) Immunostaining for Osterix in day 5 bone marrow stromal cultures resulted in nuclear localized staining (green) that was restricted to ∼68% of OC9+ cells (red). (<b>I–K</b>) OC9 positive and negative cells fractions were harvested by FACS after 5 days in culture. Quantitative RT-PCR revealed modest up-regulation of known perivascular stromal gene markers Angpt1, Cxcl12, and SCF in OC9+ cells.</p

<i>Osterix</i>-Cre Fate Mapping Marks Bone Marrow Perivascular Stromal Cells.

<p>Immunostaining for the endothelial cell marker, CD31 revealed that <i>OEC</i> (<b>A–C1</b>) and <i>ORt</i> (<b>D–F</b>) fate mapping does not result in Cre reporter expression in endothelial cells, but in a perivascular stromal cell population associated with vascular sinuses of the bone marrow. (<b>A, A1,</b> and <b>D</b>) Cre reporter expression (shown in red). (<b>B, B1,</b> and <b>E</b>) CD31 immunostaining (shown in green). (<b>C, C1,</b> and <b>F</b>) Overlay showing association of Cre reporter expressing cells with bone marrow vasculature.</p

Murine supraspinatus tendon injury model to identify the cellular origins of rotator cuff healing

<p><i>Purpose of this study</i>: To elucidate the origin of cell populations that contribute to rotator cuff healing, we developed a mouse surgical model where a full-thickness, central detachment is created in the supraspinatus. <i>Materials and methods</i>: Three different inducible Cre transgenic mice with Ai9-tdTomato reporter expression (PRG4-9, αSMA-9, and AGC-9) were used to label different cell populations in the shoulder. The defect was created surgically in the supraspinatus. The mice were injected with tamoxifen at surgery to label the cells and sacrificed at 1, 2, and 5 weeks postoperatively. Frozen sections were fluorescently imaged then stained with Toluidine Blue and re-imaged. <i>Results</i>: Three notable changes were apparent postoperatively. (1) A long thin layer of tissue formed on the bursal side overlying the supraspinatus tendon. (2) The tendon proximal to the defect initially became hypercellular and disorganized. (3) The distal stump at the insertion underwent minimal remodeling. In the uninjured shoulder, tdTomato expression was seen in the tendon midsubstance and paratenon cell on the bursal side in PRG4-9, in paratenon, blood vessels, and periosteum of acromion in SMA-9, and in articular cartilage, unmineralized fibrocartilage of supraspinatus enthesis, and acromioclavicular joint in AGC-9 mice. In the injured PRG4-9 and SMA-9 mice, the healing tissues contained an abundant number of tdTomato+ cells, while minimal contribution of tdTomato+ cells was seen in AGC-9 mice. <i>Conclusions</i>: The study supports the importance of the bursal side of the tendon to rotator cuff healing and PRG4 and αSMA may be markers for these progenitor cells.</p