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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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Gene expression profiles of DKK1, CYR61, CTGF, TGFBR1, COL3A1, COL1A1, ITGA2, MMP14, MMP2, TIMP2, TIMP1, TIMP3.The expression profiles of DKK1, CYR61, CTF, TGFBR, COL3A1, COL1A1 and ITGA2, MMP14, MMP2, TIMP2, TIMP1, TIMP3 up to 14 days in the gel type and sponge type scaffolds with and without platelet-derived growth factor-BB (PDGF) was measured using qPCR. (Up-regulated genes) Data shows x-fold increase of DKK1, CYR61, CTF, TGFBR, COL3A1, COL1A1 relative to gel type scaffold without PDGF at the corresponding time point (green line). (Down-regulated genes) Data shows x-fold decrease of ITGA2, MMP14, MMP2, TIMP2, TIMP1, TIMP3 relative to gel type scaffolds without PDGF at the corresponding time point (green line). Data points represent 5 samples
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pone-0112680-g006: Gene expression profiles of DKK1, CYR61, CTGF, TGFBR1, COL3A1, COL1A1, ITGA2, MMP14, MMP2, TIMP2, TIMP1, TIMP3.The expression profiles of DKK1, CYR61, CTF, TGFBR, COL3A1, COL1A1 and ITGA2, MMP14, MMP2, TIMP2, TIMP1, TIMP3 up to 14 days in the gel type and sponge type scaffolds with and without platelet-derived growth factor-BB (PDGF) was measured using qPCR. (Up-regulated genes) Data shows x-fold increase of DKK1, CYR61, CTF, TGFBR, COL3A1, COL1A1 relative to gel type scaffold without PDGF at the corresponding time point (green line). (Down-regulated genes) Data shows x-fold decrease of ITGA2, MMP14, MMP2, TIMP2, TIMP1, TIMP3 relative to gel type scaffolds without PDGF at the corresponding time point (green line). Data points represent 5 samples

Mentions: To reveal which genes are up-regulated and down-regulated by the scaffold in the early phase of population, we performed gene array analysis of the samples collected at day 1 (Table 1). To include the cells that are migrating towards the scaffold we isolated RNA from the sponge type scaffolds and the gel type scaffolds with the surrounding “active zone” as indicated (Figure 1A). We found that CTGF, CYR61, DKK1 were among the genes that were up-regulated 2-fold or more in the sponge type scaffolds compared to the gel type scaffolds. HAS2 and IGF2 were increased in the sponge type scaffolds with PDGF compared to the sponge type scaffolds. The gene array data was deposited into the National Center for Biotechnology Information Gene Expression Omnibus database with the accession numbers GSE61314, GSM1501968, GSM1501969, GSM1501970, and GSM1501971. Based on these results we proceeded to evaluate the gene regulation kinetics of the genes regulated in the scaffolds for up to 14 days using qPCR. In addition, we chose to analyze genes related to migration, proliferation, and matrix interaction (CCND1, ITGA2, ITGB1, PDGFRA, PDGFRB), matrix production (COLA1, COL3A1, FN1, POSTN, TNC), remodeling (MMP1, MMP2, MMP3, MMP14, TIMP1, TIMP2, TIMP3), and cell signaling factors (CCL2, IL-11). These genes were chosen based on their signal strength of above 1000, as determined by gene array, and their relevance in oral soft tissue. We found that the area under the curve above 1 for DKK1, CYR61, CTGF, TGFBR1, COL3A1, COL1A1 were increased (Table 1) over time indicating up-regulation. Further analysis revealed that CRY61, DKK1, and TGFBR1 showed an early response whereas COL1A1 and COL3A1 showed a later response in the model (Figure 6, see also Table S6 and S7 for the corresponding data). CRY61 expression peaked at day 1 whereas DKK1 and TGFBR1 peaked at day 4 in sponge type scaffolds with and without PDGF, and gel type scaffolds with PDGF relative to the gel type scaffolds. COL3A1 expression peaked at day 10 in sponge type scaffold, sponge type scaffold with PDGF, and gel type scaffolds with PDGF relative to the gel type scaffolds. In the sponge type scaffolds, CTGF peaked at day 4 whereas CTGF expression increased though day 14 in the sponge type scaffolds with PDGF. The CTGF expression in the gel type scaffolds with PDGF was not modulated. These results were consistent with the area under the curve analysis.


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)

Gene expression profiles of DKK1, CYR61, CTGF, TGFBR1, COL3A1, COL1A1, ITGA2, MMP14, MMP2, TIMP2, TIMP1, TIMP3.The expression profiles of DKK1, CYR61, CTF, TGFBR, COL3A1, COL1A1 and ITGA2, MMP14, MMP2, TIMP2, TIMP1, TIMP3 up to 14 days in the gel type and sponge type scaffolds with and without platelet-derived growth factor-BB (PDGF) was measured using qPCR. (Up-regulated genes) Data shows x-fold increase of DKK1, CYR61, CTF, TGFBR, COL3A1, COL1A1 relative to gel type scaffold without PDGF at the corresponding time point (green line). (Down-regulated genes) Data shows x-fold decrease of ITGA2, MMP14, MMP2, TIMP2, TIMP1, TIMP3 relative to gel type scaffolds without PDGF at the corresponding time point (green line). Data points represent 5 samples
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4232419&req=5

pone-0112680-g006: Gene expression profiles of DKK1, CYR61, CTGF, TGFBR1, COL3A1, COL1A1, ITGA2, MMP14, MMP2, TIMP2, TIMP1, TIMP3.The expression profiles of DKK1, CYR61, CTF, TGFBR, COL3A1, COL1A1 and ITGA2, MMP14, MMP2, TIMP2, TIMP1, TIMP3 up to 14 days in the gel type and sponge type scaffolds with and without platelet-derived growth factor-BB (PDGF) was measured using qPCR. (Up-regulated genes) Data shows x-fold increase of DKK1, CYR61, CTF, TGFBR, COL3A1, COL1A1 relative to gel type scaffold without PDGF at the corresponding time point (green line). (Down-regulated genes) Data shows x-fold decrease of ITGA2, MMP14, MMP2, TIMP2, TIMP1, TIMP3 relative to gel type scaffolds without PDGF at the corresponding time point (green line). Data points represent 5 samples
Mentions: To reveal which genes are up-regulated and down-regulated by the scaffold in the early phase of population, we performed gene array analysis of the samples collected at day 1 (Table 1). To include the cells that are migrating towards the scaffold we isolated RNA from the sponge type scaffolds and the gel type scaffolds with the surrounding “active zone” as indicated (Figure 1A). We found that CTGF, CYR61, DKK1 were among the genes that were up-regulated 2-fold or more in the sponge type scaffolds compared to the gel type scaffolds. HAS2 and IGF2 were increased in the sponge type scaffolds with PDGF compared to the sponge type scaffolds. The gene array data was deposited into the National Center for Biotechnology Information Gene Expression Omnibus database with the accession numbers GSE61314, GSM1501968, GSM1501969, GSM1501970, and GSM1501971. Based on these results we proceeded to evaluate the gene regulation kinetics of the genes regulated in the scaffolds for up to 14 days using qPCR. In addition, we chose to analyze genes related to migration, proliferation, and matrix interaction (CCND1, ITGA2, ITGB1, PDGFRA, PDGFRB), matrix production (COLA1, COL3A1, FN1, POSTN, TNC), remodeling (MMP1, MMP2, MMP3, MMP14, TIMP1, TIMP2, TIMP3), and cell signaling factors (CCL2, IL-11). These genes were chosen based on their signal strength of above 1000, as determined by gene array, and their relevance in oral soft tissue. We found that the area under the curve above 1 for DKK1, CYR61, CTGF, TGFBR1, COL3A1, COL1A1 were increased (Table 1) over time indicating up-regulation. Further analysis revealed that CRY61, DKK1, and TGFBR1 showed an early response whereas COL1A1 and COL3A1 showed a later response in the model (Figure 6, see also Table S6 and S7 for the corresponding data). CRY61 expression peaked at day 1 whereas DKK1 and TGFBR1 peaked at day 4 in sponge type scaffolds with and without PDGF, and gel type scaffolds with PDGF relative to the gel type scaffolds. COL3A1 expression peaked at day 10 in sponge type scaffold, sponge type scaffold with PDGF, and gel type scaffolds with PDGF relative to the gel type scaffolds. In the sponge type scaffolds, CTGF peaked at day 4 whereas CTGF expression increased though day 14 in the sponge type scaffolds with PDGF. The CTGF expression in the gel type scaffolds with PDGF was not modulated. These results were consistent with the area under the curve analysis.

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