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Cell population kinetics of collagen scaffolds in ex vivo oral wound repair.

Agis H, Collins A, Taut AD, Jin Q, Kruger L, Görlach C, Giannobile WV - PLoS ONE (2014)

Bottom Line: Absolute cell number and distance was higher in sponge type scaffolds when compared to gel type scaffolds, in particular during the first week of observation.Gene expression dynamics revealed the induction of key genes associated with the generation of oral tissue.DKK1, CYR61, CTGF, TGFBR1 levels were increased and integrin ITGA2 levels were decreased in the sponge type scaffolds compared to the gel type scaffold.

View Article: PubMed Central - PubMed

Affiliation: Periodontics and Oral Medicine, School of Dentistry, University of Michigan, Ann Arbor, Michigan, United States of America; Department of Conservative Dentistry and Periodontology, Medical University of Vienna, Vienna, Austria; Austrian Cluster for Tissue Regeneration, Vienna, Austria.

ABSTRACT
Biodegradable collagen scaffolds are used clinically for oral soft tissue augmentation to support wound healing. This study sought to provide a novel ex vivo model for analyzing healing kinetics and gene expression of primary human gingival fibroblasts (hGF) within collagen scaffolds. Sponge type and gel type scaffolds with and without platelet-derived growth factor-BB (PDGF) were assessed in an hGF containing matrix. Morphology was evaluated with scanning electron microscopy, and hGF metabolic activity using MTT. We quantitated the population kinetics within the scaffolds based on cell density and distance from the scaffold border of DiI-labled hGFs over a two-week observation period. Gene expression was evaluated with gene array and qPCR. The sponge type scaffolds showed a porous morphology. Absolute cell number and distance was higher in sponge type scaffolds when compared to gel type scaffolds, in particular during the first week of observation. PDGF incorporated scaffolds increased cell numbers, distance, and formazan formation in the MTT assay. Gene expression dynamics revealed the induction of key genes associated with the generation of oral tissue. DKK1, CYR61, CTGF, TGFBR1 levels were increased and integrin ITGA2 levels were decreased in the sponge type scaffolds compared to the gel type scaffold. The results suggest that this novel model of oral wound healing provides insights into population kinetics and gene expression dynamics of biodegradable scaffolds.

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Related in: MedlinePlus

Fluorescence images of human gingival fibroblasts populating the gel type and the sponge type scaffolds.Fluorescence microscopy shows DiI-labeled human gingival fibroblasts populating the gel type and the sponge type scaffolds. Images from days 1, 4, and 14 are shown. White arrows indicate the cells at the migration front. The border of the scaffoldss is indicated by a red line. The white boxes show the regions of interest (0.75 mm2), starting at the border of the scaffolds, where quantification of cell numbers and migration distance was performed. The region was 0.5 mm high and 1.5 mm wide. The photos were colorized using Adobe Illustrator. The white bar represents 500 µm.
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pone-0112680-g002: Fluorescence images of human gingival fibroblasts populating the gel type and the sponge type scaffolds.Fluorescence microscopy shows DiI-labeled human gingival fibroblasts populating the gel type and the sponge type scaffolds. Images from days 1, 4, and 14 are shown. White arrows indicate the cells at the migration front. The border of the scaffoldss is indicated by a red line. The white boxes show the regions of interest (0.75 mm2), starting at the border of the scaffolds, where quantification of cell numbers and migration distance was performed. The region was 0.5 mm high and 1.5 mm wide. The photos were colorized using Adobe Illustrator. The white bar represents 500 µm.

Mentions: The DiI-labled hGFs populating the sponge type and gel type scaffolds were examined by bright field and fluorescence microscopy utilizing a Leica DMIRB inverted microscope (Leica Microsystems GmbH, Wetzlar, Germany) at the Microscopy and Image Analysis Laboratory (University of Michigan, Ann Arbor, MI, USA). At days 1, 4, 7, 10, and 14 of culture, photos were taken with an Olympus DP-30 B&W camera (Olympus, Central Valley, PA, USA) using the 5x objective with bright field and fluorescence. The border between BME and scaffolds was identified in the bright field images and transferred onto the fluorescence images using Adobe Illustrator (Adobe Systems, Inc., San Jose, CA USA). Two rectangular regions of interest, 500 µm in width and 1500 µm in length, were defined to measure the cells populating the scaffolds and were positioned perpendicular to the defect margin (Figure 2). The coded images were analyzed by an independent, blinded examiner (AC) using ImageJ (National Institutes of Health, USA). With ImageJ the labeled cells within the regions of interest were counted and their distance to the border of the scaffolds was measured. In total, seven sponge type scaffolds and six gel type scaffolds were evaluated per group. The data are given as mean ± standard deviation.


Cell population kinetics of collagen scaffolds in ex vivo oral wound repair.

Agis H, Collins A, Taut AD, Jin Q, Kruger L, Görlach C, Giannobile WV - PLoS ONE (2014)

Fluorescence images of human gingival fibroblasts populating the gel type and the sponge type scaffolds.Fluorescence microscopy shows DiI-labeled human gingival fibroblasts populating the gel type and the sponge type scaffolds. Images from days 1, 4, and 14 are shown. White arrows indicate the cells at the migration front. The border of the scaffoldss is indicated by a red line. The white boxes show the regions of interest (0.75 mm2), starting at the border of the scaffolds, where quantification of cell numbers and migration distance was performed. The region was 0.5 mm high and 1.5 mm wide. The photos were colorized using Adobe Illustrator. The white bar represents 500 µm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4232419&req=5

pone-0112680-g002: Fluorescence images of human gingival fibroblasts populating the gel type and the sponge type scaffolds.Fluorescence microscopy shows DiI-labeled human gingival fibroblasts populating the gel type and the sponge type scaffolds. Images from days 1, 4, and 14 are shown. White arrows indicate the cells at the migration front. The border of the scaffoldss is indicated by a red line. The white boxes show the regions of interest (0.75 mm2), starting at the border of the scaffolds, where quantification of cell numbers and migration distance was performed. The region was 0.5 mm high and 1.5 mm wide. The photos were colorized using Adobe Illustrator. The white bar represents 500 µm.
Mentions: The DiI-labled hGFs populating the sponge type and gel type scaffolds were examined by bright field and fluorescence microscopy utilizing a Leica DMIRB inverted microscope (Leica Microsystems GmbH, Wetzlar, Germany) at the Microscopy and Image Analysis Laboratory (University of Michigan, Ann Arbor, MI, USA). At days 1, 4, 7, 10, and 14 of culture, photos were taken with an Olympus DP-30 B&W camera (Olympus, Central Valley, PA, USA) using the 5x objective with bright field and fluorescence. The border between BME and scaffolds was identified in the bright field images and transferred onto the fluorescence images using Adobe Illustrator (Adobe Systems, Inc., San Jose, CA USA). Two rectangular regions of interest, 500 µm in width and 1500 µm in length, were defined to measure the cells populating the scaffolds and were positioned perpendicular to the defect margin (Figure 2). The coded images were analyzed by an independent, blinded examiner (AC) using ImageJ (National Institutes of Health, USA). With ImageJ the labeled cells within the regions of interest were counted and their distance to the border of the scaffolds was measured. In total, seven sponge type scaffolds and six gel type scaffolds were evaluated per group. The data are given as mean ± standard deviation.

Bottom Line: Absolute cell number and distance was higher in sponge type scaffolds when compared to gel type scaffolds, in particular during the first week of observation.Gene expression dynamics revealed the induction of key genes associated with the generation of oral tissue.DKK1, CYR61, CTGF, TGFBR1 levels were increased and integrin ITGA2 levels were decreased in the sponge type scaffolds compared to the gel type scaffold.

View Article: PubMed Central - PubMed

Affiliation: Periodontics and Oral Medicine, School of Dentistry, University of Michigan, Ann Arbor, Michigan, United States of America; Department of Conservative Dentistry and Periodontology, Medical University of Vienna, Vienna, Austria; Austrian Cluster for Tissue Regeneration, Vienna, Austria.

ABSTRACT
Biodegradable collagen scaffolds are used clinically for oral soft tissue augmentation to support wound healing. This study sought to provide a novel ex vivo model for analyzing healing kinetics and gene expression of primary human gingival fibroblasts (hGF) within collagen scaffolds. Sponge type and gel type scaffolds with and without platelet-derived growth factor-BB (PDGF) were assessed in an hGF containing matrix. Morphology was evaluated with scanning electron microscopy, and hGF metabolic activity using MTT. We quantitated the population kinetics within the scaffolds based on cell density and distance from the scaffold border of DiI-labled hGFs over a two-week observation period. Gene expression was evaluated with gene array and qPCR. The sponge type scaffolds showed a porous morphology. Absolute cell number and distance was higher in sponge type scaffolds when compared to gel type scaffolds, in particular during the first week of observation. PDGF incorporated scaffolds increased cell numbers, distance, and formazan formation in the MTT assay. Gene expression dynamics revealed the induction of key genes associated with the generation of oral tissue. DKK1, CYR61, CTGF, TGFBR1 levels were increased and integrin ITGA2 levels were decreased in the sponge type scaffolds compared to the gel type scaffold. The results suggest that this novel model of oral wound healing provides insights into population kinetics and gene expression dynamics of biodegradable scaffolds.

Show MeSH
Related in: MedlinePlus