Effects of platelet-rich plasma on human gingival fibroblast proliferation and migration in vitro

Abstract Objective This study evaluated the influence of platelet-rich plasma (PRP) on the behaviour of human gingival fibroblasts (hGFs), including fibroblast proliferation, migration and colony formation. Methods PRP was obtained from the human peripheral blood of a healthy volunteer and then was diluted into platelet concentrations of 1%, 2% and 5%. The proliferation of hGFs was determined by two methods: (1) Cell-number counting with a haemocytometer method at days 1, 3, 5 and 7; (2) Colony-forming unit-fibroblast (CFU-F) assay at 2 weeks. The migration of hGFs was evaluated with scratch assay, then recorded digital images were analysed by Image-Analysis J 1.51j8 software to compare the remaining artificial wound areas between PRP groups at 0, 24 and 48 hours. Results All hGFs that were cultivated in media with 1%, 2% and 5% PRP showed their ability to proliferate and migrate. Cell numbers incubated with 1% PRP increased significantly during the first three days and peaked at day 5, tending to be similar to their proliferation in complete medium. With concentrations of 2% and 5% PRP, hGFs outgrew and peaked at day 3, which was faster than with those in medium with 1% PRP. Especially, hGFs in the group 5% PRP proliferated with higher cell numbers than those in the other remaining groups at day 3. The hGF colony number that was formed in the group 5% PRP was significantly higher than those in the groups 1% and 2% PRP. Scratch assay showed hGFs in the groups 2% and 5% PRP almost filled the artificial wound and migrated more effectively than in the group 1% PRP at 24 hours, which was significant. Conclusion In this study, perhaps the medium with 5% PRP is the dominant option, promoting the abilities of hGFs to heal wounds, because of its fast and effective impact on cell proliferation, colony formation and migration.

A convenient method that can help attract not only PGFs, but also epithelial growth factors, vascular endothelial growth factors, insulin-like growth factor, basic fibroblast growth factor and liver cell growth factor is using autogenous platelets. Platelet-rich plasma (PRP), the first generation of platelet gel used in periodontal regeneration therapy, was defined as using numbers of platelets 3-4 times higher than their standard in the body. Because of the high concentration of platelets in PRP, it leads to a higher number of growth factors available in wound tissue, which could activate cells to generate efficiently. 18,19 Therefore, PRP therapy points in a very positive direction for periodontal regeneration with its fundamental role in promoting soft tissue healing.
In human gingiva, fibroblasts are the most common type of connective tissue cell. The estimation is that 1 cm 3 gingival connective tissue has about 200 million fibroblasts, accounting for about 5% in volume. Fibroblasts are mesenchymal cells, which are responsible for producing most of the extracellular matrix tissue. This is very important in tissue repair and wound healing. Fibroblasts in different tissues have their own specific gene expression, growth and mobility characteristics. Due to their distinctive location and origin, gingival fibroblasts have their specific reactions to each wound, allowing them to cope optimally with the special conditions of oral and gingival lesions. 2,7,15 Biologic features of in vitro human gingival fibroblasts (hGFs) depend on the PRP concentration in the medium; however, increasing the PRP concentration did not result in increasing cell numbers. 6,8,14 Some studies showed that medium with a high concentration of PRP resulted in pH changes that negatively affected cell growth potential. Many studies in the last 5 years focused on the function of PRP at low concentration in comparison with those at high concentration. 6

Subculture of hGFs
Gingival tissue was collected from a healthy patient who underwent gingivectomy for aesthetic reasons. and 100 IU/mL penicillin (Sigma-Aldrich, MO, USA) at 37°C, 5% CO 2 until 80% confluence]. We used hGFs at passage 4 in our study.

PRP preparation
Human peripheral blood was collected from a healthy, non-smoking volunteer aged 20 to 30 years old, then was put into 3 test tubes (8.5 mL/tube) and centrifuged immediately at 2,000 rpm for 10

Effect of PRP on hGF proliferation
In general, in different cultured media of this experiment, hGFs were able to proliferate.
The line of the group 1% PRP in Figure 1 was similar to those in the control groups, which showed hGF cell numbers increased from day 1 to day 3, then peaked at day 5. At day 3 and day 5, the quantity of cells in the group 1% PRP was significantly lower than in the positive control group and higher than in the negative one (p<0.05).
In the groups 2% and 5% PRP, hGFs had a different growth tendency compared with the group 1% PRP: cell number peaked at day 3, then decreased at day 5 ( Figure 1).
At day 3, in the group 5% PRP, the quantity of hGFs was significantly higher than in the group 2% PRP (p<0.001) and 1% PRP (p<0.001). Cell number in the groups 1% and 2% PRP were not significantly different from each other (p=0.63). At day 5, cell number of the group 1% PRP continuously augmented and was significantly higher than of 2% PRP (p=0.003) and 5% PRP (p=0.004). There was no difference in cell number between the groups 2% and 5% PRP  At 24 h, no significant difference was found between the groups 2% and 5% PRP (p=0.99). The cell-free area percentage in the groups 2% PRP (29.02%) and 5% PRP (27.73%) were significantly lower than in the group 1% PRP (p=0.001). At 48 h, although the cellfree area percentage in the group 1% PRP (24.37%) was higher than in the groups 2% PRP (13.93%) and 5% PRP (15.94%), this difference was not statistically significant (p>0.05) (Figure 7).

Discussion
In recent years, the use of PRP became a popular therapy in order to provide growth factors for wounds and promote tissue regeneration. It had lots of advantages in comparison with other therapies, because PRP originated from autologous blood. The substantial advantages were its simplicity, safety and reasonable cost. Although the application of PRP was still being studied, some researchers showed evidence of a significant capacity for the increase of bone and soft tissue formation. 12,17 However, some other researchers did not find any good points. 8,16 Therefore, the effect of PRP on wound healing has been discussed until now. In this study, we used activated PRP, in which Ca 2+ was added to release growth factors inside of platelets.
Heretofore, some recent studies have shown a PRP dose-dependent proliferation increase in the cells under investigation, 11,21 with increasing PRP  The period of 7 days was suitable for observing cell proliferation to its threshold and then contractionary phase due to the inhibition.
Our study showed that in 5% PRP-supplemented medium, hGFs had the best proliferation at day 3 when the cell number was significantly higher than in other media. This result was consistent with Tavassoli with 1%, 2% and 5% PRP, the group 5% PRP seemed to be the best for hGFs to survive and self-renew.
In scratch assay, we have to control the proliferation of cells as an exclusion factor, therefore, cell migration results can be properly determined. The limitations of our study were that we did not use any growth inhibiting medications, such as aphidicolin, or reduce the serum concentration in cultivated media to restrict cell proliferation, as some methods to control cell number increase in previous studies. 10  2% and 5% demonstrated their abilities to proliferate and migrate. However, these abilities depended on the dose of PRP in media. In the proliferation experiment using the haemocytometer cell counting method, 5% PRP in medium supported hGFs to proliferate and peak in a short time; moreover, cell number in this group was significantly higher than in other groups at day 3.
Also, 5% PRP was also the best concentration in the current study to stimulate hGFs to self-renew and form colonies. In scratch assay, 2% PRP and 5% PRP were the best factors to augment the migration of hGFs in comparison with 1% PRP and positive control groups.
Eventually, this study suggested medium with 5% PRP was the dominant option, promoting the abilities of hGFs to heal wounds.

Conflicts of interest
No conflicts of interest are declared.