Experimental model for collagen estimation in cell culture

Keira, Sidney Mamoru; Ferreira, Lydia Masako; Gragnani, Alfredo; Duarte, Ivone da Silva; Barbosa, José Ignacio Rodríguez

doi:10.1590/s0102-86502004000700005

Cited by 21 publications

(22 citation statements)

References 5 publications

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“…Total soluble collagen contents in the culture medium were assessed by a modified Sirius Red Dye assay . This method is based on the selective binding property of the anionic Sirius‐red dye to the [Gly–X–Y] tripeptide end sequence of intact mammalian collagen.…”

Section: Methodsmentioning

confidence: 99%

“…Total soluble collagen contents in the culture medium were assessed by a modified Sirius Red Dye assay. 21 This method is based on the selective binding property of the anionic Sirius-red dye to the [Gly-X-Y] tripeptide end sequence of intact mammalian collagen. Briefly, 100 mL diluted cell culture medium supernatants collected at various time points in culture, were mixed with 1 mL 0.1% Sirius Red Dye (Sigma) in acetic acid for 30 min (culture medium not exposed to cells was used as blank).…”

Section: Extracellular Collagen Assaymentioning

confidence: 99%

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Proliferation and osteogenic response of MC3T3‐E1 pre‐osteoblastic cells on porous zirconia ceramics stabilized with magnesia or yttria

Hadjicharalambous

Mygdali

Prymak

et al. 2015

J Biomedical Materials Res

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Dense zirconia ceramics are used in bone applications due to their mechanical strength and biocompatibility, but lack osseointegration. A porous interface in contact with bone tissue may lead to better bone bonding but the biological properties of porous zirconia are not widely explored. The present study focuses on the manufacturing of an yttria- (YSZ) and a magnesia-stabilized (MgSZ) porous zirconia, and on their in vitro biological investigation. The sintered ceramics had similar characteristics of porosity, pore size and interconnectivity. Their elastic moduli and compressive strength values were within the range of the values of human cortical bone. MC3T3-E1 pre-osteoblasts were used to investigate the proliferation, alkaline phosphatase (ALP) activity, collagen deposition and expression profile of four genes involved in bone metabolism of cells on porous ceramics. Scanning electron and fluorescence microscopy were employed to visualize cell morphology and growth. Pre-osteoblasts adhered well on both ceramics but cell numbers on YSZ were higher. Cells exhibited an increase in ALP activity and collagen deposition after 14 days on both MgSZ and YSZ, with higher levels on YSZ. Real-time quantitative polymerase chain reaction (qPCR) showed that the expression of bone sialoprotein (Bsp) and collagen type I (col1aI) were significantly higher on YSZ. No significant differences were found in their ability to regulate the early gene expression of Runx2 and Alp. Nevertheless, the biomineralized calcium content was similar on both ceramics after 21 days, indicating that despite chemical differences, both scaffolds direct the pre-osteoblasts toward a mature state capable of mineralizing the extracellular matrix.

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Section: Methodsmentioning

confidence: 99%

Section: Extracellular Collagen Assaymentioning

confidence: 99%

Proliferation and osteogenic response of MC3T3‐E1 pre‐osteoblastic cells on porous zirconia ceramics stabilized with magnesia or yttria

Hadjicharalambous

Mygdali

Prymak

et al. 2015

J Biomedical Materials Res

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“…Sirius red assay 1.25 · 10 5 HMSC were plated in a six-well plate and cultured with 1.25 · 10 5 HUVEC for 48 h. 5 · 10 4 separated HMSC were plated in each well of a 12-well plate for another 12 h. Collagen concentration in a cultured medium was measured by the Sirius Red assay as described [18]. Briefly, the culture medium was collected and incubated with 25% ammonium sulfate solution overnight at 4°C.…”

Section: Lentivirus Expression and Infectionmentioning

confidence: 99%

Endothelial Cells Direct Mesenchymal Stem Cells Toward a Smooth Muscle Cell Fate

Lin

Lilly

2014

Stem Cells and Development

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“…Collagen was estimated by the picrosirius red procedure [40], using an aliquot of the tissue homogenates prepared for Western blotting, mixing it with Sirius Red saturated in picric acid incubated for 30 minutes, and centrifuged at 15,000 ¥ g for 5 minutes, to pellet the collagen. This pellet is rinsed once with 0.1M HCl to remove excess dye, centrifuged again, and the pellet is extracted in 0.5M NaOH, clarified, and measured spectrophotometrically at 550 nm.…”

Section: Determinations In Tissue Homogenatesmentioning

confidence: 99%

Separate or Combined Treatments with Daily Sildenafil, Molsidomine, or Muscle‐Derived Stem Cells Prevent Erectile Dysfunction in a Rat Model of Cavernosal Nerve Damage

Kovanecz

Rivera

Nolazco

et al. 2012

The Journal of Sexual Medicine

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Introduction. Long‐term daily administration of phosphodiesterase type 5 (PDE5) inhibitors in the rat prevents or reverses corporal veno‐occlusive dysfunction (CVOD) and smooth muscle cell (CSMC) loss and fibrosis, in both aging and bilateral cavernosal nerve resection (BCNR) models for erectile dysfunction. In the aging rat model, corporal implantation of skeletal muscle‐derived stem cells (MDSC) reverses CVOD. Nitric oxide (NO) and cyclic guanosine monophosphate can modulate stem cell lineage. Aim. To investigate in the BCNR model the effects of sildenafil at lower doses, alone or in combination with MDSC or the NO donor molsidomine, on CVOD and the underlying corporal histopathology. Main Outcomes Measures. CVOD, histological, and biochemical markers in rat corporal tissue. Methods. Rats subjected to BCNR were maintained for 45 days either untreated, or received sildenafil in the water or retrolingually at 10, 2.5, and 1.25 mg/kg/day (medium, low, and very low doses), or intraperitoneal molsidomine, or MDSC implantation into the corpora cavernosa separately or in combination. Cavernosometry evaluated CVOD. Histopathology was assessed on penile sections by Masson trichrome, immunohistochemistry for α‐smooth muscle actin (ASMA), or immunofluorescence for neuronal nitric oxide synthase (nNOS)/neurofilament 70, and in fresh tissue by Western blot for various markers and picrosirius red for collagen. Results. All treatments normalized erectile function (drop rate), and most increased the CSMC/collagen ratio and ASMA expression in corporal tissue sections, and reduced collagen content in the penile shaft. MDSC also increased nNOS and brain‐derived neurotrophic factor. The combination treatment was not superior to MDSC or sildenafil given alone, and upregulated PDE5. Conclusions. Lowering the dose of a continuous long‐term sildenafil administration still maintained the prevention of CVOD in the BCNR rat previously observed, but it was less effective on the underlying histopathology. As in the aging rat model, MDSC also counteracted CVOD, but supplementation with very low‐dose sildenafil did not improve the outcome. Kovanecz I, Rivera S, Nolazco G, Vernet D, Segura D, Gharib S, Rajfer J, and Gonzalez‐Cadavid NF. Separate or combined treatments with daily sildenafil, molsidomine, or muscle‐derived stem cells, prevent erectile dysfunction in a rat model of cavernosal nerve damage. J Sex Med 2012;9:2814–2826.

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Experimental model for collagen estimation in cell culture

Cited by 21 publications

References 5 publications

Proliferation and osteogenic response of MC3T3‐E1 pre‐osteoblastic cells on porous zirconia ceramics stabilized with magnesia or yttria

Proliferation and osteogenic response of MC3T3‐E1 pre‐osteoblastic cells on porous zirconia ceramics stabilized with magnesia or yttria

Endothelial Cells Direct Mesenchymal Stem Cells Toward a Smooth Muscle Cell Fate

Separate or Combined Treatments with Daily Sildenafil, Molsidomine, or Muscle‐Derived Stem Cells Prevent Erectile Dysfunction in a Rat Model of Cavernosal Nerve Damage

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