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Accelerated wound closure in vitro by fibroblasts from a subgroup of cleft lip/palate patients: role of transforming growth factor-α.

Beyeler J, Schnyder I, Katsaros C, Chiquet M - PLoS ONE (2014)

Bottom Line: Wound closure rate showed highly significant differences between fibroblast strains.Transforming growth factor-α mRNA was significantly up-regulated in the "fast" group. 5 ng/ml transforming growth factor-α added to the culture medium increased the wound closure rate of cleft lip/palate strains from the "intermediate" migratory group to the level of the "fast", but had no effect on the latter group.Conversely, antibody to transforming growth factor-α or a specific inhibitor of its receptor most effectively reduced the wound closure rate of "fast" cleft lip/palate strains.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthodontics and Dentofacial Orthopedics, School of Dental Medicine, University of Bern, Bern, Switzerland.

ABSTRACT
In a fraction of patients surgically treated for cleft lip/palate, excessive scarring disturbs maxillary growth and dento-alveolar development. Since certain genes are involved in craniofacial morphogenesis as well as tissue repair, a primary defect causing cleft lip/palate could lead to altered wound healing. We performed in vitro wound healing assays with primary lip fibroblasts from 16 cleft lip/palate patients. Nine foreskin fibroblast strains were included for comparison. Cells were grown to confluency and scratch wounds were applied; wound closure was monitored morphometrically over time. Wound closure rate showed highly significant differences between fibroblast strains. Statistically, fibroblast strains from the 25 individuals could be divided into three migratory groups, namely "fast", "intermediate", and "slow". Most cleft lip/palate fibroblasts were distributed between the "fast" (5 strains) and the "intermediate" group (10 strains). These phenotypes were stable over different cell passages from the same individual. Expression of genes involved in cleft lip/palate and wound repair was determined by quantitative PCR. Transforming growth factor-α mRNA was significantly up-regulated in the "fast" group. 5 ng/ml transforming growth factor-α added to the culture medium increased the wound closure rate of cleft lip/palate strains from the "intermediate" migratory group to the level of the "fast", but had no effect on the latter group. Conversely, antibody to transforming growth factor-α or a specific inhibitor of its receptor most effectively reduced the wound closure rate of "fast" cleft lip/palate strains. Thus, fibroblasts from a distinct subgroup of cleft lip/palate patients exhibit an increased migration rate into wounds in vitro, which is linked to higher transforming growth factor-α expression and attenuated by interfering with its signaling.

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Small effect of PDGF-CC on wound closure by fibroblasts in vitro.Boxplots represent the RWC in scratch wound assays at 24 h (*p<0.05, **p<0.01) of “fast” (A) and “intermediate” (B) CLP strains in 0.3% FCS/DMEM in the absence (control; Ctrl) or the presence of PDGF-CC diluted at either 5 ng/ml (PC5), 20 ng/ml (PC20) or 50 ng/ml (PC50). Micrographs show representative examples at 0 and 24 h in the absence (Ctrl) or presence of PDGF-CC (P20; P50). Area of scale bar corresponds to 10% RWC or 0.32mm2.
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pone-0111752-g006: Small effect of PDGF-CC on wound closure by fibroblasts in vitro.Boxplots represent the RWC in scratch wound assays at 24 h (*p<0.05, **p<0.01) of “fast” (A) and “intermediate” (B) CLP strains in 0.3% FCS/DMEM in the absence (control; Ctrl) or the presence of PDGF-CC diluted at either 5 ng/ml (PC5), 20 ng/ml (PC20) or 50 ng/ml (PC50). Micrographs show representative examples at 0 and 24 h in the absence (Ctrl) or presence of PDGF-CC (P20; P50). Area of scale bar corresponds to 10% RWC or 0.32mm2.

Mentions: As we have shown in Fig. 3, PDGFC and PDGFRB were also differentially expressed between migratory groups, suggesting a possible contribution of cellular PDGF-CC to wound closure in scratch assays. Since PDGFC was more highly expressed in the “intermediate” group, however, we expected little effect of exogenously added PDGF-CC in terms of changes in the RWC. To minimize a possible contribution of PDGF-BB from serum, the following experiments were conducted in low-serum conditions. PDGF-CC was added to cultures at 5, 20, and 50 ng/ml in 0.3% FCS/DMEM. For the “intermediate” group, the highest concentration somewhat increased the speed of wound closure (∼1.2-fold, p<8×10−03; Fig. 6B); lower concentrations in the physiological range did not have any effect. For the “fast” group, small increases of the RWC were measured at 20 ng/ml (∼1.2-fold, p<0.02) and 50 ng/ml (∼1.1-fold, p<0.03), but not at 5 ng/ml. In summary, high concentrations of PDGF-CC appeared to slightly stimulate the speed of wound closure for both “intermediate” and “fast” migratory strains, but effects were small compared to TGF-α even at 5 ng/ml.


Accelerated wound closure in vitro by fibroblasts from a subgroup of cleft lip/palate patients: role of transforming growth factor-α.

Beyeler J, Schnyder I, Katsaros C, Chiquet M - PLoS ONE (2014)

Small effect of PDGF-CC on wound closure by fibroblasts in vitro.Boxplots represent the RWC in scratch wound assays at 24 h (*p<0.05, **p<0.01) of “fast” (A) and “intermediate” (B) CLP strains in 0.3% FCS/DMEM in the absence (control; Ctrl) or the presence of PDGF-CC diluted at either 5 ng/ml (PC5), 20 ng/ml (PC20) or 50 ng/ml (PC50). Micrographs show representative examples at 0 and 24 h in the absence (Ctrl) or presence of PDGF-CC (P20; P50). Area of scale bar corresponds to 10% RWC or 0.32mm2.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4216129&req=5

pone-0111752-g006: Small effect of PDGF-CC on wound closure by fibroblasts in vitro.Boxplots represent the RWC in scratch wound assays at 24 h (*p<0.05, **p<0.01) of “fast” (A) and “intermediate” (B) CLP strains in 0.3% FCS/DMEM in the absence (control; Ctrl) or the presence of PDGF-CC diluted at either 5 ng/ml (PC5), 20 ng/ml (PC20) or 50 ng/ml (PC50). Micrographs show representative examples at 0 and 24 h in the absence (Ctrl) or presence of PDGF-CC (P20; P50). Area of scale bar corresponds to 10% RWC or 0.32mm2.
Mentions: As we have shown in Fig. 3, PDGFC and PDGFRB were also differentially expressed between migratory groups, suggesting a possible contribution of cellular PDGF-CC to wound closure in scratch assays. Since PDGFC was more highly expressed in the “intermediate” group, however, we expected little effect of exogenously added PDGF-CC in terms of changes in the RWC. To minimize a possible contribution of PDGF-BB from serum, the following experiments were conducted in low-serum conditions. PDGF-CC was added to cultures at 5, 20, and 50 ng/ml in 0.3% FCS/DMEM. For the “intermediate” group, the highest concentration somewhat increased the speed of wound closure (∼1.2-fold, p<8×10−03; Fig. 6B); lower concentrations in the physiological range did not have any effect. For the “fast” group, small increases of the RWC were measured at 20 ng/ml (∼1.2-fold, p<0.02) and 50 ng/ml (∼1.1-fold, p<0.03), but not at 5 ng/ml. In summary, high concentrations of PDGF-CC appeared to slightly stimulate the speed of wound closure for both “intermediate” and “fast” migratory strains, but effects were small compared to TGF-α even at 5 ng/ml.

Bottom Line: Wound closure rate showed highly significant differences between fibroblast strains.Transforming growth factor-α mRNA was significantly up-regulated in the "fast" group. 5 ng/ml transforming growth factor-α added to the culture medium increased the wound closure rate of cleft lip/palate strains from the "intermediate" migratory group to the level of the "fast", but had no effect on the latter group.Conversely, antibody to transforming growth factor-α or a specific inhibitor of its receptor most effectively reduced the wound closure rate of "fast" cleft lip/palate strains.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthodontics and Dentofacial Orthopedics, School of Dental Medicine, University of Bern, Bern, Switzerland.

ABSTRACT
In a fraction of patients surgically treated for cleft lip/palate, excessive scarring disturbs maxillary growth and dento-alveolar development. Since certain genes are involved in craniofacial morphogenesis as well as tissue repair, a primary defect causing cleft lip/palate could lead to altered wound healing. We performed in vitro wound healing assays with primary lip fibroblasts from 16 cleft lip/palate patients. Nine foreskin fibroblast strains were included for comparison. Cells were grown to confluency and scratch wounds were applied; wound closure was monitored morphometrically over time. Wound closure rate showed highly significant differences between fibroblast strains. Statistically, fibroblast strains from the 25 individuals could be divided into three migratory groups, namely "fast", "intermediate", and "slow". Most cleft lip/palate fibroblasts were distributed between the "fast" (5 strains) and the "intermediate" group (10 strains). These phenotypes were stable over different cell passages from the same individual. Expression of genes involved in cleft lip/palate and wound repair was determined by quantitative PCR. Transforming growth factor-α mRNA was significantly up-regulated in the "fast" group. 5 ng/ml transforming growth factor-α added to the culture medium increased the wound closure rate of cleft lip/palate strains from the "intermediate" migratory group to the level of the "fast", but had no effect on the latter group. Conversely, antibody to transforming growth factor-α or a specific inhibitor of its receptor most effectively reduced the wound closure rate of "fast" cleft lip/palate strains. Thus, fibroblasts from a distinct subgroup of cleft lip/palate patients exhibit an increased migration rate into wounds in vitro, which is linked to higher transforming growth factor-α expression and attenuated by interfering with its signaling.

Show MeSH
Related in: MedlinePlus