Platelet-Poor Plasma as a Supplement for Fibroblasts Cultured in Platelet-Rich Fibrin.

The aim of this study was to evaluate the proliferation and adhesion of mesenchymal cells (3T3/NIH) in Dulbecco's Modified Eagle Medium(DMEM) supplemented with Platelet-Poor Plasma (PPP) in aPlatelet-Rich Fibrin (PRF) scaffold. Human blood was obtained and processed in a centrifuge considering the equation G=1.12xRx(RPM/1000)2 to obtain PRF and PPP.Cell adhesion and maintenance analyses were performed by MTTassays in a 96 well plate withsupplemented DMEM: PPP (90:10) for 24 hours. Besides, the PRF was deposited in a 48 well plate and 10x104 cells were seeded above each PRF (n=3) with 800µl of DMEM: PPP (90:10) and cultured for 7 days. Histological analysis and the immunohistochemical staining for Vimentin were performed. Results were analyzed by one-way ANOVA in Stata12®. A significant decrease (p<0.05) of cells adhesion in relationship to FBSwas observed. However, a similar ability of cell-maintenance for PPP 10% was observed (P>0.05). Fibroblasts culture for 7 days in PRF supplemented with PPP 10% was possible, showing positive staining for Vimentin. Therefore, PPP cell supplementation decreased the initial adhesion of cells but was able to maintain the proliferation of adhered cells and able to support their viability in PRF.It seems that this method has many clinical advantagessince it provides an autologous and natural scaffold with their respective supplement for cell culture by only one process, without using xenogeneic compounds. This could improve the potential of clinical translational therapies based on the use of PRF cultured cells, promoting the regenerative potential for future use in medicine and dentistry.

Introduction

The clinical use of Platelet-rich fibrin (PRF) in regenerative therapies has shown the capacity to improve the biological outcomes in periodontal () and endodontic ()treatments, actingprincipally as analogous to the extracellular matrix. In this way,arecent randomized clinical trial evaluating the revascularization of immature permanent teeth showed an improvement ofbiological response in groups treated with PRFinstead of using conventional revascularization or usingPlatelet-RichPlasma (PRP) (). Althoughthe PRF seems to increase the cells response in revascularization, the tissue formed in this way is more similar to repaired tissue than to regenerated tissue becauseit does not present dentin deposition (). Thus,the use of mesenchymal cells (MC) seeded in the PRF could improve the biological capacity fortissue regeneration. However, the use of mesenchymal cells seeded and cultured in this natural scaffold is a difficult task, principally due to use of xenogeneic agents used incell culture and expansion (, ).

The identification of optimal condition for mesenchymal cells culture comprises a challenge for the transition of cell-based therapies from the bench to the bedside ().To be clinically applied, MC must be isolated and expanded in vitro (, ). Ranges of culture media, including Dulbecco’s Modified Eagle Medium (DMEM), have been employed for the in vitro maintenance of MC. To allow the cells remaining in physiological conditions, the culture medium should be supplemented with a complex mixture of growth factors, proteins, carbohydrates and cytokines ()[REMOVED HYPERLINK FIELD]. Fetal Bovine Serum (FBS) is the solution commonly applied for this purpose, despite present risks associated with transmission of zoonosis and pathogens (). Besides, animal (xenogeneic) proteins that are present in FBS can be internalized into the human cells byactivatingantigenic response post-implantation. In addition, FBS-proteins remain internalized incells cytoplasm evenafter successive washings () andmay causechanges in the surface markers, which couldinducealterationsin cells biology ().

Venous blood derivatives (VBD)have been developed as an alternative to traditional supplements to overcome the risks related to xenogeneic serums. Venous blood derivatives such as Platelet Rich Plasma have been widely studied, proving to be able to maintainMS culture as well as FBS (, ).The use of platelet concentrates in tissue regeneration can provide a more effective healing and can accelerate the repair process (). However, the protocols for obtaining the PRP are complex and there is a need for incorporation of chemicals,which are indispensable for proper PRP processing (). Unlike the PRP,the PRFhas been shown to be efficient for endodontic and periodontal regeneration and can be obtained by a simple protocol, without any addition of chemicals to human blood ().

During blood centrifugation, in order to obtainPRF, platelets are activated with a significant growth factor release - Transforming Growth Factor Beta I (TGF-β1), Insulin-Like Growth Factor I (IGF-I), Platelet-Derived Growth Factor Beta (PDGF-β) - which are sequestered in the fibrin network during the polymerization process. Recent studies (, ) have shown that the supernatant resulting from the obtainedPRF, the Platelet-Poor Plasma (PPP), could contain some grow factors in its composition. In addition, the PPP is able to induce the osteoblastic differentiation of stem cells from periodontal ligament in vitro () and the repair of bone defects in vivo (). To the best of our knowledge, there have been no reports evaluating PPPas DMEM supplementationas an alternative to FBS. Besides, after the PRF has been obtained, the PPP is generally discarded. Thus, the aim of this study was to evaluate the proliferation and adhesion of mesenchymal cells (3T3/NIH) in DMEM supplemented with Platelet-poor plasma in the PRF scaffold.

Materials and Methods

Study design:In the first part of the study, theinitial cell adhesion and proliferation in a two-dimension (2D) environmentwas evaluated, where the cells were supplemented with PPP. In the second part, the maintenance of mesenchymal cells seeded in the PRF and supplemented with PPPwas evaluated.

PRF and PPP Obtaining: Venous blood was donated by own researchersafter approval by the institution's Research Ethics Committee Number 62282216.8.0000.5318. The samples were handled immediately after blood collection under sterile conditions and biosecurity to prevent contamination in a laminar flow hood. The protocol developed by Choukroun et al. ()was applied for PRF isolation. Such a protocol relies just on centrifugation, considering the calculation of the force of gravity(G Force) producedon blood samples - G-Force = 1.12 x. Radius x (RPM / 1000): to achieve a resulting G-Force equal to 400. Thus,the blood samples were centrifuged (1,500 RPM) for 10 minutes at room temperature. After centrifugation, the portion corresponding to PPP was gently pipetted and transferred into 2 ml cryogenic vials and frozen immediately in ultra-freezer (-80C °).

Cell Culture: Fibroblast 3T3/NIH wascultured in DMEM (Cultilab®) supplemented with FBS (Cultilab®) 10%. A 75cm3 culture flask containing cells wastransferred to an incubator (37°C, 5% CO2). After reaching fibroblasts sub confluency (80%),theywerewashed withphosphate buffered saline (PBS) (Gibco®), in order to remove cell-metabolites. Subsequently, 5 ml of 0.25% trypsin/EDTA (Gibco®) has been applied on cells for 5 minutes at 37°C. For trypsin inactivation, 5 ml of standard culture media has been used. The cell-suspension was placed in 15 ml falcon-like tubes and centrifuged for 5 minutes under 1000 rpm (G-force =180). Thus, the supernatant was removed, leaving just the cell pellet. The cells were suspended in 3 ml of Standard mediawhereof 20 µL were removed for cell-counting in a hemocytometer. After counting, 2x104 cells were plated (200μl DMEM supplemented with FBS or PPP) per well in a 96 well plate.The groups (n=8) were comprised by the following supplements: DMEM: PPP (90:10) and DMEM: FBS (90:10) as the positive control; DMEM (100%) was the negative control.

Cell Adhesion Assay: Just after the addition of different supplements in the 96 well plates, cells were incubated for 24 hours. After the incubation period, DMEM+supplements wereremoved from the plate and the wells were washed with PBS. DMEM, with respective supplements,have been deposited in each well (200μl), now with the addition of MTT - (3- (4, 5-dimethylthiazol-2-yl) -2, 5 -diphenyl tetrazolium) - (0.5mg/ml) (Sigma Aldrisch®) and maintained in contact with the cells for 4h (37 ° C and 5% CO2). Post incubation, the medium was aspirated and formazan crystals weresuspended in 200μL of 10% dimethyl sulfoxide (DMSO)for 15 minutes.Then, the plate wasplaced on a shaker for 5 minutes(150 rpm). The results were assessed by spectrophotometry (Universal ELISA reader - wavelength of 540 nm), where the absorbance values were considered ​​as an indicator for cell proliferation.

Cell Maintenance Assay: To evaluate the cell maintenance, 2x104 cells were plated in a 96 well plate. All the groups were initially supplemented with FBS 10% to promote the initial adhesion of cells with the same supplementation (FBS). After 24 hours of cell adhesion, DMEM+FBS were removedfrom the plate and washed with 20μl of PBS. Then, the media was changed in the adhered-cells for DMEM:PPP (90:10) and maintained for more 24 hours. DMEM: FBS (90:10) was the positive control and DMEM (100%) the negative control. The MTT assay was carried as described above.

Cell Culture in PRF Supplemented with PPP:The PRF was obtained following the Choukrum’s protocol and deposited in a 48 well plate. Then, 10x104 cells were seeded above each of the PRF scaffolds (n=3) with 800 µl of DMEM: PPP (90:10%). The cells were cultured in a controlled environment (37°C and 5% CO2) for 7 days.The medium was changed every two days, and the PRF was washed with PBS between medium changes. The groups were fixed in formalin (4%) for 24 hours andembedded in paraffin forhistological analysis. Histological sections were performed in the long axe of the PRF. Hematoxylin-eosin stainingwas performed and evaluated under optical microscope.

Immunohistochemical Analysis: To identify the seeded fibroblasts into PRF and differentiate them from blood cells, immunohistochemical staining for Vimentin has been performed (Vimentin Immunohistology Kit Sigma-Aldrich®).

Statistical Analysis: The resultant absorbance values presented normal distribution, which wasparametrically analyzed through the one-way analysis of variance (One-way ANOVA) followed by Bonferroni complementary test. A type I error, α error of 5%, occurred. The statistical analysis was performedusing theStata 12® software.

Results

To evaluate the influence of PPP on the cell adhesion, the cells were immediately seeded in the wells with the PPP. Thus, a significant decrease (p<0.05) of cells adhesion after 24 hour in relationship to the group supplemented with FBS (Figure 1)was observed. However, it was more elevatedin the cells with DMEM (100%) (p<0.05) showing that some cells remained viable and adhered. To evaluate the cell maintenance,the cells were maintained for 24 hours in the wells to provide similar cell adhesion.After this, the cells received the PPP. Therefore, a similar ability of cell-maintenance for PPP in adhered cells without statisticaldifference (P>0.05) (Figure 2) was observed.

Figure 1

Cell adhesion of PPP 10%

Figure 2

Cell maintenance of PPP 10%

In the second part of the study, the cell culture in PRF supplemented with PPPwas evaluated.After 7 days of culture,the fibroblasts cultured in PRF and supplemented with PPP showed an expressive number of cells distributed in the PRF. In hematoxylin-eosin staining (Figure 3) it was possible to observe the cells into the fibers of PRF, principally permeating the region of clotted blood. Figure 4 shows the positive fibroblaststaining for Vimentin in a significate number of cells, confirming to be fibroblasts. Besides, the cells which were not stained for vimentin (the blood cells)were also maintained into the PRF. Thus, the PPP was also able to maintain the blood cells that remained in the PRF.

Figure 3

Fibroblasts cultured for 7 days in PPF supplemented with PPP 10%

Figure 4

Vimentin Immunohistochemically in PRF supplemented with PPP 10%

Discussion

The PPP and the PRF used in this study were obtained following the single protocol proposed by Choukroun et al. (). Such aprotocol results in a three phase-composed suspension,from which the PRF comprises the middle fraction and the red blood cells the lower portion (). The supernatant, known as PPP, comprises the upper fraction, which is discarded after the PRF has been obtained, despitethe literature reporting that the PPP possessesa growth factor profile similar to PRF (, ). In this context, this study was the first one to obtain the PRF used as natural scaffold and the use of PPP as nutritional supplement to cells culture in PRF in a single protocol. Thus, PPP used as nutritional supplement for 3T3/NIH cultivation in the PRF showed theability to maintain the cellular viability. Besides, blood cells trapped in the PRF during the centrifugation were maintained in the scaffold during this period.

The Choukrum’s protocol has been developed to obtain an autologous biomaterial which is able to induce tissue healing after surgical procedures (). For many years,the PRP has been studied and applied for repair of bone and periodontal defects (, ) and recently it has beenapplied in revascularization endodontic treatments (). However, xenogeneic agents, such as bovine thrombin,are required to obtain the PRP in order to induce coagulation and, consequently, growth factors release from the platelets (). Besides, xenogeneic agents could pose risks to patients’health due to their ability to generate antibodies against factor V and XI, which could cause coagulopathies (, ). The PRF is rich in growth factors, platelets and fibrin,thus having high potential for clinical application (, ). However, the study of PPP, which isobtained of PRF, has been poorly investigated even though ithas a high range of important biomolecules (, ). The results of this study demonstrate that PPP 10%provided similar cell-maintenance in 2D environment,which enables their useas cell-supplement in PRF, althoughthe initial adhesion in 2D has been decreased in the PPP group.

The decrease of adhesion in cells supplemented with VBD was discussed in few studies (, ). The VBD-supplements seem to decrease the expression of protein adhesion bycells supplemented with human serum and human platelet lysate (, ). The microarray assay showed a decrease of 90 genes correlated with the cell adhesion (). In this way, a decrease of cell adhesion in PPP supplement can be explained. However, after cell adhesion, a similar ability to FBS in the ability to maintain viable cells for PPP 10%was shown. Thus, the PPP at 10% seems to be an alternative viable to replace the fetal bovine serumin regenerative therapies. VBDhaveshownto improve the MC potential for translational cell-based therapies, since they allow the obtaining of all components (cells and growth factors) from the patient's body,thus reducing immunological problems (). Besides, PRF could act as a natural scaffold receiving a nutritional PPP supplementation instead of xenogeneic agents such as FBS. Thus, the patient's blood would act as source for a natural scaffold and nutritional growth factors supplementation, indispensable for MC maintenance and expansion. The present resultsshow the possibility of blood to provide a material able to serve as scaffold and supplement by the simple step-centrifugation. The vimentin staining was used to identify the fibroblast seeded in PRF, confirming the capability to adhere and be maintained into the natural scaffold using the PPP 10% as supplement tosubstitute FBS 10%. A significant amount of fibroblast cells was stained by vimentin, while the blood cells trapped in PRF during the centrifugation were not stained by vimentin, showing the possibility of PPP 10% in maintaining the viability of those human cells. The PPP provides a significant amount of platelet-derived growth factor (PDGF-AB), transforming growth factor-β1(TGF-β1) and other bioactive molecules () (, ) and this can explain the results that showed that 10% PPP provided similar cell proliferation when compared to control group.

Few studies have used concentrated blood in order to supplement cell culture and, in addition, other nutrients were added to blood concentrates to maintain the cell viability. Isaac et al. ()carried out a filtration after the addition of 10% human plasma in DMEM;aPRF fraction has been applied as an additional supplement. DMEM-Low Glucose medium supplemented with 10% autologous blood plasma (20 ng/ml):β fibroblast growth factor (βFGF) and endothelial growth factor(EGF) plus L-glutamine has, also, been applied in the study of Lin et al. (), which hasobtained good results with blood concentrates.However,it is impossible to individually observethe actual role of blood concentrated in MC proliferation. Besides, there are studies evaluating methods basedon platelet membrane disruption by freeze-thaw cycles to improve the potential supplementation. However,such atechnique requires the use of heparin to prevent coagulation of the concentrate in the culture medium (). In this context, a recent study evaluated PPP, and other VBD, combined with 10 ng/ml recombinant human epidermal growth factor (rhEGF) in adipogenic stem cell (). In this way, similar results have been observed regarding the proliferation and differentiation ability of MC supplemented with blood derivatives (PRP, PPP and human serum) compared to FBS. The addition of exogenous growth factors to PPP has provided satisfactory results in adipocyte stem cells.However, the PPP alone seems to provide a sufficient nutritional supplementation compared to FBS.

Although the method investigated in this study hasthe disadvantage because it requires blood collectedfrom the patient, it is considered to be less invasive than techniques using grafts from donor sites. Besides, the use of this method seems to be clinically advantageous since it provides an autologous and natural scaffold with their respective supplement for cell culture in only one procedure, without using xenogeneic compounds. This could improve the potential of clinical translational therapies based on the use of PRF cultured cells, promoting the regenerative potential for future use in several areas of medicine and dentistry.

Conclusions

The supplementation of 3T3/NIH cells with Platelet-Poor Plasma decreased the initial cell adhesion but was able to maintain the cell proliferation similar to the Fetal Bovine Serum. Besides, the cell viability in PRF with PPP used as supplement was ensured. Thus, it was possible to obtain a natural scaffold and the cell supplements from the blood through a single centrifugation step for use in regenerative therapies.