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Biological responses of three-dimensional cultured fibroblasts by sustained compressive loading include apoptosis and survival activity.

Kanazawa T, Nakagami G, Minematsu T, Yamane T, Huang L, Mugita Y, Noguchi H, Mori T, Sanada H - PLoS ONE (2014)

Bottom Line: Compared to the control, the compressed samples demonstrated that apoptosis was induced in a time- and load- dependent manner; vinculin and stress fiber were scarce; HSP90α, CD44, HAS2, and COX2 expression was upregulated; and the concentrations of HSP90α, hyaluronan (HA), and prostaglandin E2 (PGE2) were increased.In addition, the gene expression of antiapoptotic Bcl2 was significantly increased in the compressed samples compared to the control.These results suggest that compressive loading induces not only apoptosis but also survival activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Gerontological Nursing/Wound Care Management, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

ABSTRACT
Pressure ulcers are characterized by chronicity, which results in delayed wound healing due to pressure. Early intervention for preventing delayed healing due to pressure requires a prediction method. However, no study has reported the prediction of delayed healing due to pressure. Therefore, this study focused on biological response-based molecular markers for the establishment of an assessment technology to predict delayed healing due to pressure. We tested the hypothesis that sustained compressive loading applied to three dimensional cultured fibroblasts leads to upregulation of heat shock proteins (HSPs), CD44, hyaluronan synthase 2 (HAS2), and cyclooxygenase 2 (COX2) along with apoptosis via disruption of adhesion. First, sustained compressive loading was applied to fibroblast-seeded collagen sponges. Following this, collagen sponge samples and culture supernatants were collected for apoptosis and proliferation assays, gene expression analysis, immunocytochemistry, and quantification of secreted substances induced by upregulation of mRNA and protein level. Compared to the control, the compressed samples demonstrated that apoptosis was induced in a time- and load- dependent manner; vinculin and stress fiber were scarce; HSP90α, CD44, HAS2, and COX2 expression was upregulated; and the concentrations of HSP90α, hyaluronan (HA), and prostaglandin E2 (PGE2) were increased. In addition, the gene expression of antiapoptotic Bcl2 was significantly increased in the compressed samples compared to the control. These results suggest that compressive loading induces not only apoptosis but also survival activity. These observations support that HSP90α, HA, and, PGE2 could be potential molecular markers for prediction of delayed wound healing due to pressure.

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Related in: MedlinePlus

Loading apparatus to apply sustained compressive loading to cells seeded-collagen sponge.In this representation A indicates an indenter (diameter: 10 mm), B the weights, C a stainless steel plate (thick: 5 mm), D a 12-well plate lid, E culture medium, and F a fibroblast-seeded collagen sponge sample (diameter: 5 mm and thick: 3 mm).
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pone-0104676-g001: Loading apparatus to apply sustained compressive loading to cells seeded-collagen sponge.In this representation A indicates an indenter (diameter: 10 mm), B the weights, C a stainless steel plate (thick: 5 mm), D a 12-well plate lid, E culture medium, and F a fibroblast-seeded collagen sponge sample (diameter: 5 mm and thick: 3 mm).

Mentions: Collagen sponge samples were precultured for 24 h and subjected to sustained compressive loading under 5% CO2 at 37°C by using a custom-built loading apparatus (Fig. 1). The loading apparatus applied compression to the samples in a 12-well plate with stainless steel indenters, using a 5-mm thick stainless steel plate on top of the 12-well plate to stabilize the indenter. Various weights can be placed on top of these indenters to apply specified compression to the samples. Pressures of 0, 50, 100, or 200 mmHg, by following the report of Swain [22], were applied on samples for 2, 4, or 6 h. The sample with no treatment (0 h–0 mmHg) was analyzed as the baseline. Experiments were repeated 5 times.


Biological responses of three-dimensional cultured fibroblasts by sustained compressive loading include apoptosis and survival activity.

Kanazawa T, Nakagami G, Minematsu T, Yamane T, Huang L, Mugita Y, Noguchi H, Mori T, Sanada H - PLoS ONE (2014)

Loading apparatus to apply sustained compressive loading to cells seeded-collagen sponge.In this representation A indicates an indenter (diameter: 10 mm), B the weights, C a stainless steel plate (thick: 5 mm), D a 12-well plate lid, E culture medium, and F a fibroblast-seeded collagen sponge sample (diameter: 5 mm and thick: 3 mm).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4125229&req=5

pone-0104676-g001: Loading apparatus to apply sustained compressive loading to cells seeded-collagen sponge.In this representation A indicates an indenter (diameter: 10 mm), B the weights, C a stainless steel plate (thick: 5 mm), D a 12-well plate lid, E culture medium, and F a fibroblast-seeded collagen sponge sample (diameter: 5 mm and thick: 3 mm).
Mentions: Collagen sponge samples were precultured for 24 h and subjected to sustained compressive loading under 5% CO2 at 37°C by using a custom-built loading apparatus (Fig. 1). The loading apparatus applied compression to the samples in a 12-well plate with stainless steel indenters, using a 5-mm thick stainless steel plate on top of the 12-well plate to stabilize the indenter. Various weights can be placed on top of these indenters to apply specified compression to the samples. Pressures of 0, 50, 100, or 200 mmHg, by following the report of Swain [22], were applied on samples for 2, 4, or 6 h. The sample with no treatment (0 h–0 mmHg) was analyzed as the baseline. Experiments were repeated 5 times.

Bottom Line: Compared to the control, the compressed samples demonstrated that apoptosis was induced in a time- and load- dependent manner; vinculin and stress fiber were scarce; HSP90α, CD44, HAS2, and COX2 expression was upregulated; and the concentrations of HSP90α, hyaluronan (HA), and prostaglandin E2 (PGE2) were increased.In addition, the gene expression of antiapoptotic Bcl2 was significantly increased in the compressed samples compared to the control.These results suggest that compressive loading induces not only apoptosis but also survival activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Gerontological Nursing/Wound Care Management, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

ABSTRACT
Pressure ulcers are characterized by chronicity, which results in delayed wound healing due to pressure. Early intervention for preventing delayed healing due to pressure requires a prediction method. However, no study has reported the prediction of delayed healing due to pressure. Therefore, this study focused on biological response-based molecular markers for the establishment of an assessment technology to predict delayed healing due to pressure. We tested the hypothesis that sustained compressive loading applied to three dimensional cultured fibroblasts leads to upregulation of heat shock proteins (HSPs), CD44, hyaluronan synthase 2 (HAS2), and cyclooxygenase 2 (COX2) along with apoptosis via disruption of adhesion. First, sustained compressive loading was applied to fibroblast-seeded collagen sponges. Following this, collagen sponge samples and culture supernatants were collected for apoptosis and proliferation assays, gene expression analysis, immunocytochemistry, and quantification of secreted substances induced by upregulation of mRNA and protein level. Compared to the control, the compressed samples demonstrated that apoptosis was induced in a time- and load- dependent manner; vinculin and stress fiber were scarce; HSP90α, CD44, HAS2, and COX2 expression was upregulated; and the concentrations of HSP90α, hyaluronan (HA), and prostaglandin E2 (PGE2) were increased. In addition, the gene expression of antiapoptotic Bcl2 was significantly increased in the compressed samples compared to the control. These results suggest that compressive loading induces not only apoptosis but also survival activity. These observations support that HSP90α, HA, and, PGE2 could be potential molecular markers for prediction of delayed wound healing due to pressure.

Show MeSH
Related in: MedlinePlus