Platelet gel stimulates proliferation of human dermal fibroblasts in vitro
M. Kra{na, D. Domanovi}, A. Tom{i~, U. [vajger, and M. Jeras
-A B S T R A C T
Background: Platelet gel is a source of many soluble proteins that can modulate tissue regeneration. Dermal fibroblasts play an important role in tissue remodeling and wound healing.
Objective: The study was performed to investigate the influence of platelet gel and its active ingredients on the proliferation of human dermal fibroblasts in vitro.
Methods: A fibroblast culture was established from a normal human skin biopsy. Platelet gel was prepared from platelet-rich plasma (PRP) following the addition of calcified thrombin solution. Cell proliferation was measured by MTT assay.
Results: We showed that platelet gel, PRP, and thrombin stimulate the proliferation of dermal fibro-blasts, and that stimulation by PRP is dose dependent.
Conclusion: Platelet gel can be used to treat chronic skin defects.
K E Y WORDS
platelet gel, platelet-rich plasma, dermal-fibroblasts, proliferation, MTT assay
Tissue repair normally begins with clot formation and platelet degranulation, leading to the release of various cytokines and coagulation factors, which modulate inflammatory response. To date, more than 30 different cytokines have been found in platelets; PDGFs, TGFs, EGF, and IGF are among those most studied (1). Platelet gel is produced by mixing two solutions: platelet-rich plasma and calcified thrombin solution. The PRP can be prepared by aphaeresis or can be separated from fresh anticoagulated blood by simple centrifugation, which concentrates platelets up to six times the baseline count in whole blood (2). The production process itself (2), as well as the addition of
calcified thrombin, activates platelets, which in turn release cytokines. At the same time, fibrinogen converts into a fibrin clot. Platelets attach to the fibrin web forming the platelet gel, which can be applied directly to injured tissue to enhance its regeneration. There are several commercially available devices that simplify the production of platelet-rich plasma from a patient's blood (Vivostat® [Vivolution A/S], SmartPReP® [Harvest Technologies], and Fibrinet® [Cascade Medical Enterprises]). Platelet gel is being used in a variety of clinical procedures: as a tissue sealant (3), as a skin healing enhancer (4), or for dental and bone regeneration (5-7). Fibrin sealant, a well-known hemostatic
Figure 1. Proliferation of fibroblasts in the presence of various components of the platelet gel (MTT assay). Concentration of platelets in PRP was 887 x 109/L; calcified thrombin (10 lU/mL); 2% FBS in the medium as a positive control; medium without any additives as a negative control (C). Results shown are the means of four parallel cultures ± SD.
Figure 2. Proliferation of fibroblasts in the presence of platelet-rich plasma (PRP). Concentration of platelets in PRP was 1416 x 109/L; 2% FBS as a positive control. Results shown are the means of four parallel cultures ± SD.
material, is also a blood-derived product that contains proteins for blood coagulation but lacks platelets and cytokines. In addition, it promotes wound healing and
skin engraftment, and serves as a scaffold for fibrin-based cultured skin substitutes (8-10).
Dermal fibroblasts are one type of essential cell in
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Figure 3. Fibroblast cultures after 4 days of incubation with 0% PRP (A), 2% FBS (B), 2% PRP (C) and 20% PRP (D). PRP contained 1416 x 109 platelets/L. The scale bars are 50 |jm.
the dermis. They play an important role in repairing skin wounds by providing growth factors and extracellular matrix proteins that attract new cells, which eventually restore the physiological continuity of the skin. When the balance between cytokine and extracellular matrix protein synthesis and degradation shifts towards degradation, skin cells are no longer able to completely restore normal skin, and the damaged tissue progresses into a chronic wound. A more advanced approach to healing chronic wounds is the application of cytokines into the poorly healing tissue, so that balance is brought back to the synthesis and cell proliferation (11, 12). Platelet gel, as a source of various cytokines, could be an option to enhance the regeneration process.
The aim of this study was to investigate the influence of platelet gel and its compounds on the proliferation of human dermal fibroblasts in a cell culture.
Materials and methods
Fibroblast culture
A normal adult human skin sample was obtained from reductive surgery. The skin was cut into small pieces, which were incubated overnight at 37 °C in a collagenase solution (200 U/ml) (GibcoBRL, Invitrogen). Subsequently, the epidermis was discarded and dermal cells were isolated from the dermal layer of the skin after another overnight digestion with collagenase. Dermal cells were subcultured in a complete fibroblast medium supplemented with insulin, bFGF, 2% FBS, gentamicin (50 pg/ml), and amphotericin B (2 pg/ml) (FGM-2, Cam-brex) at 37 °C in a humidified CO2 incubator.
and cells were incubated for 24 hours in FGM-2 without serum. The medium was refreshed before adding the platelet gel compounds. Six different growth conditions were tested in the FGM-2 medium without FBS: calcified thrombin (10 |l), PRP (10 |l), platelet gel (20 |l), frozen/thawed PRP (10 |l), FGM-2 medium (negative control), and 2% FBS (positive control). The effect of different concentrations of PRP on cell proliferation was also studied. After 4 to 5 days of incubation, the proliferation of fibroblasts was measured with an MTT assay (Sigma-Aldrich Chemie). This is a colorimetric assay that measures the chemical reduction of MTT (3-[4.5-Dimethylthiazol-2-yl]-2.5-diphenyltetrazolium bromide) into formazan, which is directly proportional to the number of viable cells in the tested culture. Cultures were incubated 2 to 3 hours in a culture medium with 0.5 mg/mL of MTT. The resulting formazan was then eluted with acidified isopropanol (0.04 N HCl in isopropanol) and the optical density was measured at 570 nm, then subtracted for the optical density at 620 nm. To obtain the optical densities of all tested media, another 24-well plate was prepared as described above, but the fibroblasts were excluded. At the end of the assay, these values were subtracted from the experimental optical densities of cell culture supernatants. Proliferation was measured in four replicates. Optical densities were expressed as the mean ± standard deviation. Differences between means were assessed by Student's i-test. A value of p < 0.05 was considered to be statistically significant. Experiments were performed four times with PRP obtained from four different donors.
Results
Platelet gel preparation
Platelet-rich plasma was prepared from venous blood obtained from healthy volunteers after obtaining their signed informed consent. Blood samples were collected in 9 mL tubes with tri-sodium citrate anticoagulant. The blood was first separated into two layers by centrifugation at 272 g for 7 min. The upper layer was collected and centrifuged at 1,288 g for 7 min. PRP was collected from the resulting 1-1.5 mL sediment, and the platelets were counted (Cell-DYN 3200, Abbott). The platelet gel was prepared by mixing equal parts of PRP and calcified thrombin 500 IU/ml in 40mM solution (Tissucol, Baxter-Hyland Immuno). The study was approved by the National Medical Ethics Committee.
Fibroblast proliferation assay
Fibroblasts were seeded in 24-well plates in 0.5 mL of the FGM-2 medium with 2% FBS. When cells reached 30% of confluent growth, the medium was washed off
The platelet-rich plasma prepared by a simple cen-trifugation process from whole blood resulted in a concentration of platelets approximately three to four times the baseline count in whole blood. All components of the platelet gel significantly stimulated the growth of fibroblasts when compared to the negative control (Figure 1; p < 0.0001). The calcified thrombin alone also showed a slightly positive effect on fibroblast growth, although the stimulation was not as strong as for PRP, frozen/thawed PRP, or platelet gel. Five different concentrations of PRP (0, 0.2, 2, 10, and 20%) were tested to determine the maximal stimulating effect on fibro-blast proliferation. Fibroblast growth was enhanced in a dose dependent manner. 20% PRP showed the strongest effect, whereas 0.2% PRP did not affect growth at all (Figure 2). All fibroblast cultures retained normal morphology. After only 4 days of incubation in the presence of 20% PRP, fibroblasts completely covered the surface of the wells, forming a dense layer of cells (Figure 3). Unfortunately we could not test higher concentrations in this experiment because all the PRP was al-
ready used. In a further experiment (data not shown), we were able to test higher concentrations of PRP, including 100% PRP, but we could not measure the control media with MTT because of the lack of PRP. Nevertheless, after 4 days of incubation we observed the highest proliferation in 100% PRP with light microscopy.
Discussion
Platelet gel is a biological source of various cytokines (13), which stimulate the proliferation of osteoblasts, gingival fibroblasts (14), mouse embryonic fibroblasts (15), stromal stem cells (16), and endothelial cells (17), and enhance healing of skin ulcers (4, 18, 19). In our study we showed that allogeneic platelet gel stimulates the proliferation of primary dermal fibroblasts in vitro. The MTT assay, which we used to measure the proliferation, is based on the reduction of MTT catalyzed mainly by mitochondrial enzymes (but also by a number of other non-mitochondrial enzymes), which are abundantly present in fibroblasts as well as in platelets (20, 21). This fact was taken into consideration by subtracting the optical densities (PRP only, without cells) from those assessed in fibroblasts containing cultures incubated with PRP. Cells were cultured in a serumfree medium to avoid the stimulative effect of FBS. We showed that at least 2% PRP (approx. 28 x 106 platelets/ mL) stimulates proliferation and that the stimulation is
R E F E R E N C E S -
dose dependent, which is consistent with findings reported by other studies (22, 16). We did not observe any cytotoxic effects of PRP on proliferation and morphology. Frozen PRP and calcified thrombin were also able to stimulate cell proliferation. Frozen/thawed PRP retained its capacity to form a clot when it was mixed with calcified thrombin. This finding could be useful in preparing PRP in advance and for storage in a freezer for multiple applications (13). It has been shown before that thrombin acts as a potent fibroblast mitogen. Experiments were conducted examining the in vitro dose dependent effects of thrombin on proliferation of human skin fibroblasts in a fibrin-sealing system (23, 24). The role of growth factors promoting tissue regeneration has been well studied (25-28). A recombinant human PDGF-BB (becaplermin) has been developed and is commercially available as Regranex®. It is being used successfully to treat chronic wounds (29, 30) and periodontal defects (31). Our study contributes to this kind of knowledge because it shows the positive effect of platelet gel on the growth of human dermal fibro-blasts in vitro.
Abbrevetation
PRP- platelet-rich plasma
MTT - (3-[4.5-Dimethylthiazol-2-yl]-2.5-diphenyltetra-zolium bromide) FBS - fetal bovine serum
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AUTHORS' Metka Krasna, PhD, Department for Blood Supply, Blood Transfusion ADDRESSES Center of Slovenia, Šlajmerjeva 6, 1000 Ljubljana, Slovenia,
E-mail: metka.krasna@ztm.si Dragoslav Domanovic, PhD, same address
Ana Tomšič, BS, Faculty of Pharmacy, Aškerčeva 7,1000Ljubljana, Slovenia Urban Švajger, BS, Tissue typing centre, Blood Transfusion Center of Slovenia, Šlajmerjeva 6, 1000 Ljubljana, Slovenia Matjaž Jeras, PhD, same address