Plasma Rich In Growth Factors PromoteGingival Tissue Regenerationby Stimulating Fibroblast Proliferationand Migration and by BlockingTransforming Growth Factor-b1-InducedMyodifferentiationEduardo Anitua,*† Marıa Troya,† and Gorka Orive†

Background: Periodontitis involves inflammation and infec-tion of the ligaments and bones that support the teeth. Gingivalfibroblasts are the most abundant cells in periodontal tissue,and they play a role in maintaining the structural integrity ofthe tissue. Plasma rich in growth factors contain a pool of pro-teins and growth factors that promote wound healing andtissue regeneration. In the present study, we evaluate the po-tential of different formulations obtained with this approachto stimulate several biologic processes involved in woundhealing, including fibroblast proliferation, migration, adhe-sion, and the autocrine release of some angiogenic factorsand extracellular matrix components. Furthermore, the abilityof this technology to prevent and inhibit transforming growthfactor b1-induced myodifferentiation was also determined.

Methods: Cell proliferation was evaluated through a colori-metric assay, cell migration was performed on culture inserts,and cell adhesion was studied through a fluorescence-basedmethod. Enzyme-linked immunosorbent assay was used todetermine some of the biomolecules released by gingival fi-broblasts. Smooth muscle actin expression was assessedthrough immunofluorescence microscopy.

Results: Results showed that plasma rich in growth factorssignificantly increased gingival fibroblast proliferation, migration,and cell adhesion on type I collagen matrix. In addition, it stimu-lated the autocrine expression of vascular endothelial growth fac-tor,hepatocytegrowth factor,andhyaluronicacid.Themyofibroblastphenotype, which is characterized by expressing a-smooth muscleactin, was inhibited and reverted by treating with this technology.

Conclusion: These findings suggest that plasma rich in growthfactors is capable of promoting regeneration of gingival connectivetissue by stimulating some of the main processes involved in woundregeneration. J Periodontol 2012;83:1028-1037.

KEY WORDS

Gingiva; myofibroblasts; platelet-rich plasma; transforminggrowth factors; wound healing.

Periodontitis is the inflammationand infection of the ligaments andbones that support the teeth.1

Periodontal regeneration is a complexprocess that involves the interactions ofat least four different cell types: alveolarbone cells, periodontal ligament cells,gingival fibroblasts, and epithelial cells.2

Gingival fibroblasts are the most abun-dant resident cells in periodontal tissueand they play a role in maintaining thestructural integrity of the tissue.3

Periodontal wound healing and re-generation processes involve a complexcascade of cellular events, includingmigration, adhesion, proliferation, dif-ferentiation, and matrix synthesis. Celladhesion and cell migration are criticalevents of this intricate process. Cellmigration requires the accurate coordi-nation of many signaling pathways toinduce polarization, adhesion to the ex-tracellular matrix (ECM) components,and actin polymerization.4 Conversely,cell adhesion is mediated by cell-surfaceintegrins that regulate many other pro-cesses, including cell growth, tissue or-ganization, cell differentiation, and cellsurvival.5

* Private practice, Vitoria, Spain.† Biotechnology Institute, Vitoria, Spain. doi: 10.1902/jop.2011.110505

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It has been reported that, during gingival woundhealing, a specific subpopulation of cells known as my-ofibroblasts is differentiated.6 The latter are responsiblefor wound contraction, an important part of wound clo-sure. However, because myofibroblasts are knownto be involved in an excessive ECM deposition, theirpersistence is associated with a variety of fibrotic andpathologic situations.7 For that reason, myofibroblasts,which are characterized by expressing a-smooth mus-cleactin (a-sma)withincytoplasmicstressfibers,8 con-tribute to the connective tissue fibrosis by increasingsynthesis and decreasing degradation of ECM pro-teins.9 It has been reported that transforming growthfactor b1 (TGF-b1), a multifunctional growth factorhighly expressed in gingival tissues subjected to inflam-mation and wound healing, is the main mediator re-sponsible for myofibroblast differentiation.10,11

Platelets mediate primary homeostasis becausethey express and release substances that promotewound healing and tissue repair.12 They contain largeamounts of biologically active proteins, and they arealso able to synthesize and release new active metab-olites.13 Over the past several years, a better under-standing of the physiologic roles of platelets in thehealing process has fueled the use of platelets as ther-apeutic tools.14-17 The potential of the plasma rich ingrowth factors technology in wound healing and tissueregeneration has been evaluated in the preclinicaland clinical settings.12,14-16 This approach providesa wide range of growth factors that mediate and accel-erate cell function, driving the tissue regeneration pro-cess. Some of these proteins include platelet-derivedgrowth factor (PDGF), TGF-b1, vascular endothelialgrowth factor (VEGF), hepatocyte growth factor (HGF),insulin-like growth factor-1 (IGF), and epidermalgrowth factor (EGF), among others.18-20 Over the pastseveral years, the potential effects of different platelet-rich plasmas on oral cells have been evaluated.21-25

Assuming that the function of gingival fibroblasts iscritical during periodontal repair, the purpose of thepresent study is to assess the potential of plasma richin growth factors technology as an innovative ap-proach for enhancing periodontal regeneration. Toaddress this issue, the effect on human gingival fibro-blast proliferation, migration, and adhesion, as well ason the secretion of VEGF, HGF, procollagen type I,and hyaluronic acid (HA) induced by plasma rich ingrowth factors, was determined. The preventive andinhibiting effects of several plasma fractions from thisautologous technology on TGF-b1-stimulated myofi-broblast differentiation were also evaluated.

MATERIALS AND METHODS

Cell IsolationPrimary cultures of human gingival fibroblasts wereestablished by the explant method. Briefly, tissue

explants were obtained from tuberosity and palatinehealthy areas of three female patients during routinesurgical procedures. Written informed consent wasobtained from all patients before the biopsy was per-formed. Explants were rinsed in phosphate-bufferedsaline (PBS) containing 50 mg/mL gentamicin and2.5 mg/mL amphotericin B,‡ minced into smaller por-tions, placed in six-well plates, and incubated in Dul-becco’s modified Eagle’s medium (DMEM)/F-12 (1:1volume)§ supplemented with 15% fetal bovine se-rum (FBS),i 2 mM glutamine,¶ 50 mg/mL gentamicin,and 2.5 mg/mL amphotericin B# in a humidified atmo-sphere at 37�C with 5% CO2. After reaching approxi-mate confluence, primary fibroblasts were detachedwith animal origin-free trypsin-like enzyme** andsubcultured. Cell viability was assessed by trypanblue dye exclusion.†† All experiments were performedusing cells from these three donors, between thefourth and the fifth passages.

Characterization of Primary Cultures of HumanGingival FibroblastsThe fibroblast-like morphology of cells isolated waschecked by phase-contrast microscopy.‡‡ Cells werecharacterized by immunofluorescence using threeantibodies against typical fibroblasts markers (type Icollagen,§§ vimentin,ii and CD90¶¶) and two other an-tibodies against typical endothelial cells and hemato-poietic progenitor cell markers (CD 105 and CD34##).

Cells were fixed for 10 minutes in 4% formaldehydefor CD 90, CD 34, CD 105, and type I collagen anti-gens and in methanol/acetic acid (3:1) for vimentinantigen. Cells for type I collagen staining were per-meabilized with 1% of a non-ionic surfactant,*** inPBS for 10 minutes. After that, cells were blocked withFBS (10% in PBS) for 30 minutes and incubated for1 hour with the primary antibodies. Next, cells were in-cubated with their appropriate secondary antibodies,goat anti-mouse immunoglobulin G (IgG) conjugatedwith a fluorochrome or goat anti-rabbit IgG conju-gated with a fluorochrome.††† Finally, the cell nucleiwere stained with blue fluorescent dye,‡‡‡ mounted,and visualized under a fluorescence microscope.§§§

‡ Sigma-Aldrich, St. Louis, MO.§ Invitrogen, Grand Island, NY.i Biochrom, Berlin, Germany.¶ Sigma-Aldrich.# Sigma-Aldrich.** Invitrogen.†† Sigma-Aldrich.‡‡ Leica DM IRB, Leica Microsystems, Wetzlar, Germany.§§ Millipore, Billerica, MA.ii Sigma-Aldrich.¶¶ BD Biosciences, San Jose, CA.## BD Biosciences.*** Triton X-100, Sigma-Aldrich.††† Alexa Fluor 488 and Alexa Fluor 594, Invitrogen.‡‡‡ Hoechst 33342, Invitrogen.§§§ Leica DM IRB, Leica Microsystems.

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Plasma Rich In Growth Factors TechnologyBecause of the great variety of platelet-rich plasmaprotocols available that may lead to different biologiceffects, we emphasize that the results obtained hereare only related to plasma rich in growth factors, thepioneering autologous technologyiii to obtain a cock-tail of proteins and growth factors and a fibrin scaffoldfrom the same patient’s blood.

Blood from one healthy young male donor was col-lected into 9-mL tubes with 3.8% (weight/volume)sodium citrate, after written informed consent was pro-vided. Samples were centrifuged at 580 · g for 8 min-utes at room temperature. The 2 mL above the buffycoat were separated and used in the different experi-ments. According to the protocol provided by the man-ufacturer,¶¶¶ the 1 mL above the buffy coat, whichpresents the highest platelet and growth factor concen-trations, was identified as fraction 3 (F3), whereas thenext 1 mL above F3 was named fraction 2 (F2). Noneof the fractions included the buffy coat. In this study,both F3 (group 1) and F2+F3 (group 2) were evalu-ated.

Platelet and leukocyte counts were performed witha hematology analyzer.### Both preparations wereincubated with preparation rich in growth factors ac-tivator**** at 37�C in glass tubes. One hour later, thesupernatants were collected by aspiration after centri-fugation at 1,000 · g for 20 minutes at 4�C. Finally, theplasma obtained from both treatment groups (F2+F3and F3) was aliquoted and stored at -80�C untiluse. Growth factors (TGF-b1, PDGF-AB, VEGF, HGF,IGF-I, and EGF) were measured in the supernatantsusing commercially available colorimetric sandwichenzyme-linked immunosorbent assay kits.††††

Cell ProliferationAs described previously,26 subconfluent cultures ofcells were detached, and cell viability was assessedby trypan blue dye exclusion. Passage 5 cells wereplated at a density of 20,000 cells/cm2 in 48-well tissue-culture plates and maintained with serum-free culturemedium for 48 hours. Then, culture medium wasreplaced by serum-free medium supplemented witheither: 1) 20% (volume/volume [v/v]) F2+F3 super-natant; or 2) 20% (v/v) F3 supernatant for 72 hoursAdditional cultures were maintained in serum-freeDMEM/F-12 supplemented with 0.2% FBS to examineconstitutive secretion (non-stimulated cells). Wells withthese three treatments and without cells were main-tained in the same conditions and used for backgroundcorrection. Cell proliferation was evaluated using theWST-1 (tetrazolium salt, (4-[3,4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzenedisulphonate)colorimetric assay.‡‡‡‡ Absorbance at 450/620 nmwas directly proportional to the number of living cellsin the culture. As an index of cell number, calibration

curves ranging from 5,000 to 80,000 cells per wellwere established using the WST-1 cell counting kit.All samples were performed in triplicate.

MigrationCell migration was performed on culture inserts,§§§§

consisting of two wells as cell growth areas (0.22cm2/well) and a cell-free gap (500 – 50 mm) betweenthem.Theculture insertswereplaced into 24-well platesandcellswere seededatadensityof30,000cells/cm2 inboth wells. They were incubated in DMEM/F-12 supple-mented with 2 mM glutamine and 50 g/mL gentamicin(referred to below as the culture medium) and with 15%FBS. After 40 hours of incubation, the inserts were re-moved and the cell-free gap was created. The platescontaining these cells were then treated with serum-freeculture medium supplemented with either 1) 20% (v/v)F2+F3 supernatant or 2) 20% (v/v) F3 supernatant for24 hours. Cells maintained in serum-free culture me-dium supplemented with 0.2% FBS were consideredas a control of non-stimulated cells.

Phase-contrast images of the gap were captured at0 (control) and 24 hours of incubation using a digitalcameraiiii coupled to an inverted microscope.¶¶¶¶ Af-ter 24 hours of incubation, cells were also nuclearstained with blue fluorescent dye#### to make cellcounting easier. Images were taken from the centralpart of the gap (·5), as representatives of the entiremigration area. Cell counting was done using imaginganalysis software.***** All samples were assayed intriplicate. Results are expressed as cell number persquare millimeter.

Cell AdhesionTissue culture 96-well optical bottom black plateswere coated with 10 mg/cm2 collagen type I††††† insterile tissue culture-grade water. Excess fluid wasremoved and plates were allowed to dry overnight.Non-specific binding sites were blocked by FBS.

Passage 5 gingival fibroblasts were seeded ontocollagen type I-coated plates at a density of 20,000cells/cm2 with: 1) 20% (v/v) F2+F3 supernatant; 2)20% (v/v) F3 supernatant; or 3) 0.2% FBS for 15 min-utes. Cell adhesion was stopped by carefully pouringoff the medium, and attached cells were quantified us-ing a cell proliferation assay.‡‡‡‡‡ Briefly, treatments

iii PRGF-Endoret Technology, BTI Biotechnology Institute, Minano,Alava, Spain.

¶¶¶ PRGF-Endoret Technology, BTI Biotechnology Institute.### Micros 60, Horiba ABX, Montpellier, France.**** PRGF-Endoret Technology, BTI Biotechnology Institute.†††† R & D Systems, Minneapolis, MN.‡‡‡‡ Roche, Basel, Switzerland.§§§§ Ibidi, Martinsried, Germany.iiii Leica Microsystems.¶¶¶¶ Leica DM IRB, Leica Microsystems.#### Hoechst 33342, Invitrogen.***** NIH Image J version 1.42, National Institutes of Health, Bethesda,

MD.††††† Sigma-Aldrich.‡‡‡‡‡ CyQUANT cell proliferation assay, Invitrogen.

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were removed and wells were washed carefully withPBS, and the microplates with the cells were frozenat -80�C until assayed. After thawing the plates atroom temperature, wells were incubated with ribonu-clease A (1.35 kU/mL) diluted in cell lysis buffer for 1hour at room temperature. Then, ·2 green fluorescentdye§§§§§/cell lysis buffer was added to each samplewell, mixed gently, and incubated for 5 minutes atroom temperature, protected from light. Sample fluo-rescence was measured with a fluorescence micro-plate reader.iiiii A deoxyribonucleic acid standardcurve ranging from 7.8 to 1,000 ng/mL was includedin all fluorescence quantifications. Cells maintainedfor 24 hours were used as a control of 100% of adhe-sion. All experiments were performed in triplicate.

Secretion of Angiogenic Factors andECM ComponentsCulture medium was collected on day 3 of treatmentfrom the cell proliferation assay, centrifuged for 10minutes at 460 · g, and stored at -80�C until assayed.The amount of HGF, VEGF,¶¶¶¶¶ procollagen typeI,##### and HA****** was determined by assay kits.All samples were performed in triplicate, and resultswere expressed as nanograms per 106 cells.

Myofibroblast DifferentiationEffect of TGF-b1 dose on a-sma expression. Pas-sage 5 fibroblasts were plated at a density of 5,000cells/cm2 in 48-well tissue-culture plates and main-tained with serum-free culture medium for 48 hours.Then, culture medium was replaced by serum-freemedium supplemented with or without TGF-b1††††††

for 3 and 5 days. In the case of cells treated with TGF-b1 for 5 days, culture medium was replaced the thirdday of treatment. Cells were incubated with TGF-b1 atconcentrations of 2.5, 5, and 10 ng/mL, and cellswithout TGF-b1 were used as control. Cells from thecontrol group were supplemented with 0.2% FBS withthe aim of maintaining cell viability. After that, cellswere fixed for 10 minutes in methanol, blocked withFBS (10% in PBS) for 30 minutes, and incubated for1 hour with mouse anti-a-sma antibody‡‡‡‡‡‡ at1:800, followed by incubation with goat anti-mouseIgG conjugated with a fluorochrome§§§§§§ at 1:100for 1 hour. Finally, nuclei were stained with blue fluo-rescent dyeiiiiii and visualized under a fluorescencemicroscope.¶¶¶¶¶¶

Myofibroblast cell counting was determined by tak-ing photographs over three random ·10 microscopicfields on each well. The digitalized images were ana-lyzed using the imaging analysis software.###### Bluedye*******-positive cells were counted to obtain to-tal cell number. Blue dye†††††††-positive and a-sma-positive cells were counted as myofibroblasts. All cellsthat showed any kind of greenish staining were con-sidered a-sma-positive cells.

Effects of plasma rich in growth factors on TGF-b1-stimulated a-sma expression. Passage 5 cellswere plated at a density of 4,000 cells/cm2 in 48-welltissue-culture plates and maintained with serum-freeculture medium for 48 hours Then, two protocols werefollowed. In the first protocol, culture medium was re-placed by serum-free medium and gingival fibroblastwere stimulated with: 1) 2.5 ng/mL TGF-b1, 2) 2.5ng/mL TGF-b1 plus 20% (v/v) F2+F3 supernatant,or 3) 2.5 ng/mL TGF-b1 plus 20% (v/v) F3 superna-tant for 3 days; in the second, cells were pretreatedwith 2.5 ng/mL TGF-b1 for 3 days. After this pretreat-ment, culture medium was replaced by serum-freemedium and supplemented with one of three treat-ments: 1) 2.5 ng/mL TGF-b1, 2) 2.5 ng/mL TGF-b1plus 20% (v/v) F2+F3 supernatant, or 3) 2.5 ng/mLTGF-b1 plus 20% (v/v) F3 supernatant for 3 days.In both protocols, a supplement of 0.2% FBS wasadded to the 2.5 ng/mL TGF-b1 treatment to maintaincell viability in the control group. Experiments wereperformed in triplicate. The immunocytochemistryand myofibroblast cell counting were performed asdescribed above.

Statistical AnalysesUnivariate analysis of variance was used to assess thepotential differences among treatments for each ex-perimental procedure according to the different celllines used. To identify the differences, post hoc anal-ysis based on Scheffe or Bonferroni tests were per-formed, as appropriate. To evaluate the differencesbetween the two treatment periods, for each of theTGF-b1 concentrations tested, the Mann-Whitney Utest was performed instead. Statistical differences be-tween groups were accepted for P values 0.05. Resultsare expressed as mean – 95% confidence intervals.

RESULTS

Cultured fibroblasts showed elongated and spindle-shaped appearance. Immunofluorescence microscopyconfirmed that fibroblast cultures were uniformly pos-itive for vimentin, CD90, and type I collagen but neg-ative for markers of hematopoietic and endothelialcells (data not shown).

Plasma rich in growth factors was prepared, and thetwo plasma groups under study (group 1, F2+F3;

§§§§§ CyQUANT GR dye, Invitrogen.iiiii Twinkle LB 970, Berthold Technologies, Bad Wildbad, Germany.¶¶¶¶¶ R & D Systems.##### Takara, Shiga, Japan.****** Corgenix, Broomfield, CO.†††††† Millipore.‡‡‡‡‡‡ Sigma-Aldrich.§§§§§§ Invitrogen.iiiiii Hoechst 33342, Invitrogen.¶¶¶¶¶¶ Leica DM IRB, Leica Microsystems.###### NIH Image J, National Institutes of Health.******* Hoechst 33342.††††††† Hoechst 33342.

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group 2, F3) were characterized. Table 1 shows plate-let counts and the concentrations of some growthfactors involved in wound healing.

Cell Proliferation and MigrationAfter 72 hours of treatment, both F2+F3 and F3 aloneexerted a similar effect on gingival fibroblasts prolif-eration. Cell proliferation was significantly higherwhen plasma rich in growth factors were used com-pared to non-stimulation. In fact, cell proliferationwas increased 2.1-fold and 2-fold for F2+F3 andF3, respectively, over controls. No significant

differences were observedamong the plasma fractions(Fig. 1).

Regarding cell migration,plasma rich in growth factorshave a stimulatory effect on themigration process of human gin-gival fibroblasts. Both fractionsexerted a statistically significantincrease compared to untreatedcells. A 24-hour exposure toF2+F3 enhanced gingival fi-broblasts migration 4.3-fold,whereas the improvement incell migration exerted by F3 was2.7-fold (Fig. 2).

Cell AdhesionThe ability of gingival fibroblaststo adhere to a collagen type Imatrix was also evaluated afterstimulation with plasma rich ingrowth factors. It was observeda statistically significant in-crease in the number of the at-tached cells after treatment withboth F2+F3 or F3 alone com-pared to the control (Fig. 3).

Secretion of Angiogenic Factors and ECMComponentsThe expression of different growth factors and ECMcomponents stimulated by different fractionsderived from plasma rich in growth factors (F2+F3and F3) in human primary gingival fibroblasts wasevaluated (Fig. 4). After 72 hours of culturingthe cells with 20% F2+F3 or 20% F3, VEGF (Fig. 4A)expression was significantly higher compared tonon-stimulated cells. Similar results were obtainedwhen HGF and HA were measured (Figs. 4B and 4D).The expression of procollagen type I was stimulated

Table 1.

Platelet and Leukocyte Count and Concentration of a Range of Growth Factorsin the Plasma Formulations

Growth Factor Concentration

Plasma

Preparation

Leukocyte

Count (·106/mL)

Platelet

Count (·106/mL)

TGF-b1

(ng/mL)

PDGF-AB

(ng/mL)

VEGF

(pg/mL)

HGF

(pg/mL)

IGF-I

(ng/mL)

EGF

(pg/mL)

F2+F3 0 232 (·1.8) 30 8 71 489 101 335

F3 0.1 338 (·2.6) 50 15 114 507 105 590

Peripheral blood contained 131 · 106 platelets/mL.

Figure 1.Effect of plasma rich in growth factors on gingival fibroblast proliferation after 72 hours. A) Phase-contrastmicroscopy revealed a higher number of cells when these were treated with any of the plasma formulations.Scale bars = 200 mm. B) Proliferation was statistically higher in gingival fibroblasts treated with both F2+F3and F3 compared to non-stimulated cells. NS = non-stimulated cells. *P <0.05, statistically significantdifferences between treated and non-stimulated cells.

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by both plasma treatments, although results were notsignificantly higher than the control samples (Fig.4C).

Myofibroblast DifferentiationTo determine the effects of three different TGF-b1concentrations on myofibroblast differentiation, theexpression of the specific myofibroblast markera-sma during two different periods was assessed. Fig-ures 5A and 5B show how TGF-b1 induced a-smaexpression in the treated cells. The percentage ofa-sma-positive cells after 3 days of TGF-b1 treatmentwas lower than after 5 days of treatment. However, dif-ferences between both treatment periods were notsignificant except for the dose of 10 ng/mL. Addition-ally, no significant differences were observed amongthe three TGF-b1 doses at either time period.

The effects of plasma fractions (F2+F3 and F3) onthe prevention of the TGF-b1-stimulated myofibro-blast differentiation were evaluated. Cells treatedwith the different plasma fractions alone were notdifferentiated into myofibroblasts (data not shown).

After 3 days of treatment with2.5 ng/mL TGF-b1, a total of30% – 8% of the fibroblastsbecome a-sma positive. How-ever, in the case of cells treatedwith either 2.5 ng/mL TGF-b1plus 20% F2+F3 supernatantor 2.5 ng/mL TGF-b1 plus20% F3 supernatant, the a-sma-positive cells were <0.1%(Fig. 5C).

Inasecondsetof experiments,all the cell groups were pre-treated with 2.5 ng/mL TGF-b1for 3 days and then cultured withthe different plasma fractions.The percentage of a-sma-posi-tive cells after pretreatment with2.5 ng/mL TGF-b1 was signifi-cantly lower in those groupsin which TGF-b1 was supple-mented with plasma fractions.Addition of both F2+F3 and F3alone to the culture dramati-cally reduced the TGF-b1-in-duced a-sma-positive cells by97% – 1% and 92% – 2%, respec-tively (Fig. 5D).

DISCUSSION

Growth factors participate in al-most all cell processes involvedin the wound-healing cascade,including the stimulation of cel-

lular division, migration, cell adhesion, differentiation,and gene expression.27 One way of delivering hun-dreds of autologous proteins and growth factorsto the wound site is the use of plasma rich in growthfactors technology. In fact, a recent systematic re-view28 has underlined the potential of these platelet-rich plasma products for oral and maxillofacial sur-gery, although the great variety of platelet-richplasma protocols available today may lead to differ-ent biologic effects.

Here, we study the effect of plasma rich in growthfactors, to obtain a cocktail of proteins and growth fac-tors and a fibrin scaffold from the same patient’sblood‡‡‡‡‡‡‡ on human gingival fibroblasts. This proce-dure consists of a 100% autologous and biocompatibleplatelet-rich plasma elaborated from patient’s bloodusing one-step centrifugation process and sodium cit-rate and calcium chloride as anticoagulant and activa-tor, respectively.12,14,16 This autologous technologycontains a moderated platelet concentration that has

Figure 2.Effect of different fractions obtained from plasma rich in growth factors on gingival fibroblast migrationafter 24 hours. A) Phase-contrast microscopy and blue dye§§§§§§§ staining showed fibroblast migrationafter 24 hours with both treatments and control when compared to the gap free of cells at the verybeginning of the migration process. Scale bars = 400 mm. B) The area covered with migrated cells werestatistically higher with both plasma treatments compared to the non-stimulation situation. NS =non-stimulated cells. *P <0.05, statistically significant difference between treated and non-stimulatedcells. †P <0.05, statistically significant differences between F2+F3 and F3.

§§§§§§§ Hoechst 33342, Invitrogen.‡‡‡‡‡‡‡ PRGF-Endoret Technology, BTI Biotechnology Institute.

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been related to optimal biologic benefit.29 Leukocytecontent has been eliminated from plasma rich in growthfactors with the aim of avoiding the proinflammatory ef-fects of the proteases and acid hydrolases contained inwhite blood cells, as well as the large amounts ofprofibrotic agents secreted by the inflammation-related lymphocytes, macrophages, and activatedmast cells.30

Results obtained in this study confirm that plasmarich in growth factors technology stimulates the essen-tial processes related to tissue regeneration in gingivalfibroblasts. In fact, when fibroblasts were stimulatedwith both fractions obtained from this autologous tech-nology, cell proliferation, cell migration, and cell adhe-sion to type I collagen were enhanced significantly.

These results are consistent with findings reportedby a number of other studies.21,22,24,31-34 This may beattributed to the combined action of several growthfactors present in the platelet-rich plasma. For exam-ple, VEGF, PDGF, and IGF are reported to stimulatecell migration, whereas TGF-b1, EGF, and againPDGF present a potent mitogen role.18,34-36

Integrins play an important role during woundhealing through their function in cell adhesion andsignaling. Cytokines and growth factors are knownto regulate integrin expression. In fact, PDGF has

been reported to stimulate a-2integrin in fibroblasts, allowingcell adhesion to a collagen ma-trix.37,38 The expression of twomain angiogenic factors suchas VEGF and HGF, as well ashyaluronic acid was also en-hanced in gingival fibroblastsafter treating with plasma richin growth factors. Of particularnote is HGF, which has beenreported to exert potent anti-fibrotic and anti-inflammatoryeffects. The latter are mediatedvia enhancing cellular nuclearfactor of k-light polypeptidegene enhancer in B-cells in-hibitor a expression, whichcontributes to nuclear factork-light-chain-enhancer of acti-vated B cells-p65 subunit re-tention in the cytosol andnucleo-cytoplasmic shuttling.39

In general, oral tissue is char-acterized by reduced contrac-tile properties, great motility,and reduced scarring.40 How-ever, a number of situations,including the administrationof several drugs, hereditary

Figure 3.Effect of different plasma rich in growth factors formulations on gingivalfibroblasts adhesion to a collagen type I matrix. When fibroblasts werestimulated with both fractions of plasma, the percentage of cell adhesionwas significantly enhanced four-fold over the control. NS = non-stimulatedcells. *P <0.05, statistically significant differences between treated andnon-stimulated cells.

Figure 4.Effect of plasma fractions on the secretion of angiogenic factors (VEGF and HGF) and ECM components(procollagen type I and HA). A) Effect on VEGF secretion. B) Effect on HGF secretion. C) Effect onprocollagen Type I secretion. D) Effect on HA secretion. NS = non-stimulated cells. *P <0.05, statisticallysignificant differences between treated and non-stimulated cells.

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gingival fibromatosis, and chronic inflammation, maylead to gingival overgrowth and differentiation, lead-ing to high levels of certain cytokines, such as TGF-b1.41 It has been reported that this factor, an impor-tant immune regulator during inflammatory response,is responsible for the activation of the profibrotic cas-cade that occurs after injuries and diseases.42,43 Infibrotic tissue, the increase of a-sma expression upre-gulates the contractile activity of myofibroblasts,

which in turn activates latentTGF-b1 from the ECM.44 Aftertissue injury, fibroblasts differ-entiate into contractile and se-cretory myofibroblasts thatcontribute to tissue repair dur-ing wound healing but that canseverely impair tissue func-tion when contraction andECM protein secretion be-come excessive. Myofibroblastsrelease angiogenic, proinf-lammatory, and profibroticfactors that may stimulate fi-brosis.40,45

Results obtained in this studyconfirm that plasma rich ingrowth factors inhibit and revertTGF-b1-induced a-sma ex-pression of fibroblasts. In fact,when cells were treated with ei-ther 2.5 ng/mL TGF-b1 plus20% F2+F3 supernatant or 2.5ng/mL TGF-b1 plus 20% F3supernatant, the percentageof a-sma-positive cells was<0.1%. Moreover, addition ofboth F2+F3 and F3 alone tothe TGF-b1-pretreated culturedramatically reduced theTGF-b1-induced a-sma-posi-tive cells by 97% – 1% and92% – 2%, respectively. There-fore, the addition of 20% ofboth plasma fractions, whichrepresented an extra additionof 6 or 10 ng/mL TGF-b1, wasnot correlated with an increasein the myodifferentiation rateof the fibroblasts. On the con-trary, this biologic phenotypewas inhibited and reverted bytreating cells with the cocktailof proteins and growth factorspresent in plasma rich in growthfactors.

The exact mechanisms be-hind the activity of plasma rich in growth factors arenot totally understood. In fact, the great range of mol-ecules involved in this autologous approach makes itextremely challenging to determine the key mole-cules and mechanisms involved in inhibiting andreversing myofibroblast differentiation. Several stud-ies, however, are shedding light on this issue. Forexample, HGF has been reported to be the maininhibitor of the TGF-b1-induced myofibroblast

Figure 5.TGF-b1-stimulated a-sma expression by human gingival fibroblasts. A and B) Effects of three differentTGF-b1 concentrations on a-sma expression by human gingival fibroblasts. A) Immunofluorescence ofgingival fibroblasts after treatment with increasing concentrations of TGF-b1 for 3 and 5 days. Scale bars =200 mm. B) After the 5-day treatment, the percentage of a-sma-positive cells was higher than after 3 days,but significant differences were only found for the dose of 10 ng/mL. *P <0.05, statistically significantdifferences for the dose of 10 ng/mL between 3- and 5-day treatments. †P <0.05, statistically significantdifferences between 0 ng/mL TGF-b1 and the rest of the concentrations in both treatment periods. C)Percentage of a-sma-positive cells after treatment with 2.5 ng/mL TGF-b1, 2.5 ng/mL TGF-b1 plus 20%F2+F3, or 2.5 ng/mL TGF-b1 plus 20% F3, for 3 days. D) Percentage of reduction of a-sma staining inpretreated cells that were treated with either TGF-b1 plus 20% F2+F3 or TGF-b1 plus 20% F3. ‡P <0.05,statistically significant differences between treatments.

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differentiation.46,47 HGF may increase the inhibitoryprotein Smad 7 whose overexpression leads to an in-hibition of TGF-b1-induced myofibroblast transfor-mation by means of decreasing Smad 2 and Smad3 phosphorylation. Furthermore, connective tissuegrowth factor, another molecule present in prepara-tion rich in growth factor, has been demonstrated tobe critical in myofibroblast differentiation.41,48-50

CONCLUSION

Although additional studies are needed to elucidatethe underlying mechanism by which this technologyregulates the biologic activity, together, these findingssuggest that plasma rich in growth factors is capableof promoting regeneration of gingival connective tis-sue by stimulating the main processes involved inwound regeneration and of providing an interestingapproach for gingival situations characterized bythe persistence of a chronic inflammation and ele-vated levels of TGF-b1.

ACKNOWLEDGMENTS

The study was funded by the Biotechnology Institute(BTI), Vitoria, Spain. Dr. Anitua is the scientific direc-tor and Dr. Orive and Ms. Troya are researchers at BTI.

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Correspondence: Eduardo Anitua, Instituto Eduardo Ani-tua, Jose Maria Cagigal 19, 01007 Vitoria, Spain. Fax: 34-945160657; e-mail: eduardoanitua@eduardoanitua.com.

Submitted August 25, 2011; accepted for publicationOctober 18, 2011.

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