research article platelet-rich fibrin promotes...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 638043 13 pageshttpdxdoiorg1011552013638043

Research ArticlePlatelet-Rich Fibrin Promotes Periodontal Regeneration andEnhances Alveolar Bone Augmentation

Qi Li12 Shuang Pan3 Smit J Dangaria1 Gokul Gopinathan1 Antonia Kolokythas4

Shunli Chu2 Yajun Geng5 Yanmin Zhou2 and Xianghong Luan1

1 UIC Brodie Laboratory for Craniofacial Genetics 801 South Paulina Chicago IL 60612 USA2Department of Implantology Stomatological Hospital Jilin University Changchun Jilin 130021 China3Department of Endodontics School of Dentistry Harbin Medical University Harbin China4Department of Oral and Maxillofacial Surgery UIC College of Dentistry Chicago IL USA5The First Hospital of Jilin University Jilin China

Correspondence should be addressed to Yanmin Zhou zhouym62126com and Xianghong Luan luanuicedu

Received 12 November 2012 Revised 2 January 2013 Accepted 4 January 2013

Academic Editor Brian L Foster

Copyright copy 2013 Qi Li et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

In the present study we have determined the suitability of platelet-rich fibrin (PRF) as a complex scaffold for periodontaltissue regeneration Replacing PRF with its major component fibrin increased mineralization in alveolar bone progenitorswhen compared to periodontal progenitors suggesting that fibrin played a substantial role in PRF-induced osteogenic lineagedifferentiation Moreover there was a 36-fold increase in the early osteoblast transcription factor RUNX2 and a 31-fold reductionof the mineralization inhibitor MGP as a result of PRF application in alveolar bone progenitors a trend not observed inperiodontal progenitors Subcutaneous implantation studies revealed that PRF readily integrated with surrounding tissues andwas partially replaced with collagen fibers 2 weeks after implantation Finally clinical pilot studies in human patients documentedan approximately 5mm elevation of alveolar bone height in tandem with oral mucosal wound healing Together these studiessuggest that PRF enhances osteogenic lineage differentiation of alveolar bone progenitors more than of periodontal progenitorsby augmenting osteoblast differentiation RUNX2 expression and mineralized nodule formation via its principal componentfibrin They also document that PRF functions as a complex regenerative scaffold promoting both tissue-specific alveolar boneaugmentation and surrounding periodontal soft tissue regeneration via progenitor-specific mechanisms

1 Introduction

Regenerative oral medicine entails the replacement of tissueslost to disease or injury with physiologically equivalentengineered tissues Often tissues in the oral cavity are ofcomplex nature with bordering mineralized and soft tis-sue components both of which harbor unique progenitorpopulations residing within specialized extracellular matrixframeworks [1 2] Mimicking such complex environmentsby using chemically homogenous scaffolds and uniformstem cell populations is often challenging Instead recentapproaches favor complex natural scaffolds that allow forrepopulation with the patientrsquos own cells thereby producingan autologous tissue-engineered organ [3]

One such complex natural scaffold ideally suited forautologous tissue regeneration is platelet-rich fibrin (PRF)

a second generation platelet concentrate developed as animprovement over the earlier introduced platelet-rich plasma(PRP) as an aid for tissue repair and regeneration [4] In con-trast to PRP which was prepared by adding bovine thrombinand anticoagulants PRF is generated from centrifuged bloodand is strictly autologous PRF predominantly consists of afibrin matrix rich in platelet and leukocyte cytokines suchas IL-1120573 -4 and -6 and growth factors such as TGF-1205731PDGF-AB and VEGF [5] Fibrin gels exploit the final stage ofthe coagulation cascade in which fibrinogen molecules self-assemble into a highly biocompatible three-dimensional fibernetwork [6]The combination of fibrins and cytokines withinPRF becomes a powerful bioscaffold with an integratedreservoir of growth factors for tissue regeneration [7] Thesuitability of PRF as a biologically active scaffold has been

2 BioMed Research International

illustrated in a number of studies revealing proliferation anddifferentiation of osteoblasts and gingival fibroblasts [8 9]Clinical studies have demonstrated that PRF promotes softtissue and bone regeneration [10 11] as well as periodontaltissue regeneration [12 13]The ability of PRF to augment andregenerate compromised tissues may be enhanced in combi-nation with bone substitutes such as Bio-Oss or autologousbone [14 15] Together these studies have established PRF as ahighly biocompatible and inductive scaffold useful for a broadrange of tissue engineering applications

In the present study we have hypothesized that PRF mayprovide a scaffold material for periodontal tissue regener-ation Earlier studies have reported the effect of PRF onperiodontal cells [16 17] Here we have compared the effectsof PRF on dental follicle periodontal ligament and alveolarbone cells [1] using microscopy as well as biological assaysfor migration proliferation mineralization and gene expres-sion We also tested PRF scaffold properties in subcutaneousimplants in nude mice Finally we report on the use of PRFas a scaffold for peri-implant alveolar bone augmentationin clinical use Together these studies for the first timecharacterize the tissue-specific biological effects of PRF as abioactive scaffold to promote periodontal soft and hard tissueregeneration

2 Materials and Methods

21 Preparation of PRF To prepare PRF 10mL of freshblood from the precaval vein of a pig was collected into10mL glass-coated tubes without anticoagulants All pigsused in this study were female with an average age of 31months (range from 29 to 35 months) The platelet countof whole pig blood was 105120583L This study was approved bythe Ethics Committee of the University of Illinois at ChicagoUSA As described in previous studies [4 18] samples wereimmediately centrifuged at 2100 rpm (approximately 400 g)for 12 minutes using a Beckman centrifuge The PRF clotswere concentrated between the red blood cell corpusclesat the bottom of the centrifuge tubes and acellular plasmacalled platelet-poor plasma (PPP) at the top of the tubesPPP was collected by pipetting the supernatant of the cen-trifuged blood preparation After PPP removal PRF clotswere mechanically separated from red blood cells and gentlycompressed using gauze to drain the remaining fluid

Reproducibility of studies was ensured by maintain-ing consistent preparation conditions for PRF membranesincluding centrifugation force and time tube size and plateletcount of pig blood and by completing blood collectionprocedures within 2 minutes For in vitro studies all cellculture wells were treated with one whole PRF clot consistingof identical amounts of fibrin platelets and white blood cellsand each PRF clot was freshly prepared from blood

22 Preparation of Conditioned Medium To prepare condi-tioned medium PRF membranes were soaked in 5mL freshDMEM medium without fetal bovine serum in 6-well cellculture plates The conditioned medium was collected every48 h and fresh medium was added into wells after collection

23 Isolation of Human Dental Progenitor Cells To generatehuman dental progenitor cells healthy human teeth (patientsranging from 12 to 15 years) were extracted for orthodonticreasons in accordance with the human subjects protocolapproved byUICrsquos Institutional Review Boards and theOfficefor the Protection Research Subjects The dental follicle (DF)was dissected from developing tooth organs and alveolarbone (AB) and periodontal ligament (PDL) were preparedfrom teeth with tooth roots already formed Mesenchymalstem cells were isolated from the dental tissues after digestionwith collagenasedispase as described before [2]

24 MTT Cell Proliferation Assay PDL DF and AB cells(104 cellswell) were seeded into 96-well plates and culturedfor 8 hours After cells were attached each well was washedtwice with phosphate-buffered saline solution (PBS) andeither PRF-conditionedmedium + 10 FBS or DMEM+ 10PPP + 10 FBS was added to the well DMEM + 10 FBSwas used as a control Prior to the termination of culture cellswere incubated inMTT solution (2120583gmL ofMTT inDMEMwith 2 FBS) for 4 hours To quantify proliferative activitythe MTT-stained cells were lysed in HCLisopropanol andthe absorbance was detected at 570 nm with backgroundsubtraction at 630 nm Cell proliferation was detected on adaily basis over the entire 7-day culture period

25 Chemotaxis Assay Cellmigration assayswere performedusing a FluoroBlok-24-multiwell insert system (DB Bio-sciences Bedford MA) PRF-conditioned medium DMEM+ 10 PPP or DMEM was added into separate wells of thelower chambers of the FluoroBlok system Serum-starvedcells (105) were seeded into each insert and allowed tomigrateto the bottomof themembrane for 12 hoursThenonmigratedcells on the upper surface of the membrane were removedwith a cotton swab and themigratory cells that were attachedto the lower surface were stained with DAPI The numbersof migrated cells per membrane were counted under a LeicaDMRX fluorescent microscope

26 Induction of Osteogenic Differentiation Alkaline Phos-phatase (ALP) Activity Assessment and Alizarin Red S Stain-ing PDL DF and AB were seeded into 6-well plates at aconcentration of 104 cellswell and cultured for 8 hours Aftercell attachment PRF membranes were added to individualwells soaked and subjected to osteogenic treatment whichincluded addition of osteogenic medium (OM) DMEM +10 PPP + 10 FBS or DMEM to each individual wellThe following four groups were employed in this study(i) PRF in DMEM with 10 FBS (ii) 10 PPP in DMEMwith 10 FBS (iii) osteogenic medium with 10 FBS and(iv) DMEM with 10 FBS To test the effect of fibrinas the major PRF component on periodontal progenitordifferentiation and mineralization fibrin was used to coatotherwise untreated cell culture dishes and compared withvacuum gas plasma-treated and plasma-untreated dishes Togenerate fibrin equal amounts of fibrinogen (100mgmL)and thrombin (500 120583mL) were mixed prior to applicationSubsequently treated cells were cultured for an additional 7

BioMed Research International 3

14 or 21 days For alkaline phosphatase activity assays cellswere washed and stained with NBTBCIP (Roche DiagnosisGmbH Mannheim Germany) after rinsing with PBS Forquantification of mineralized nodules cells were fixed withmethanol and stained with 10 alizarin red solution Min-eralized nodules were identified as red spots on the culturedish Alkaline phosphatase activity was determined after oneweek and alizarin red S staining was performed after threeweeks Wells were scanned and the integrated optical density(average intensityobject density) of the stained area wascalculated using the Image Pro Plus 60 software (MediacyChicago IL USA)

27 RNA Extraction and RT PCR Total RNAs were isolatedfrom cultured cells using the TRIZOL LS Reagent (Invitro-gen Carlsbad CA USA) according to the manufacturerrsquosinstructions Two micrograms of total extracted RNA wasapplied toward cDNA generation with the Sprint RT Com-plete kit (Clontech Mountain View CA USA) PCR primerswere designed based on EMBLGenBank searches andprimer sequences were as followsRUNX2 51015840-TTACTTACACCCCGCCAGTC-31015840 (sense) 31015840-CACTCTGGCTTTGGGAAGAG-51015840 (antisense)MGP 51015840-CCCTCAGCAGAGATGGAG AG-31015840 (sense) 31015840-GCTTCCCTATTGAGCTCGTG-51015840(antisense) 120573-ACTIN 51015840-GCA TGG GTC AGA AGG ATTCCT-3 (sense) and 31015840-TCG TCC CAG TTG GTG ACGAT-51015840 (antisense) Real-time PCR was performed usingsequence-specific SyberGreen primers and the ABI Prism7000 sequence detection system (Applied Biosystems FosterCity CA USA) Reaction conditions were as follows 2minat 50∘C (one cycle) 10min at 95∘C (one cycle) 15 sec at 95∘Cand 1min at 60∘C (40 cycles) Samples were normalized using120573-actin The analyses were performed in triplicate for threeindependent experiments to confirm reproducibility of theresults Relative expression levels were calculated using the2minusΔΔCt method [19] and values were graphed as the meanexpression level plusmn standard deviation

28 Protein Extraction and Western Blot Analysis For west-ern blot evaluation of the effect of PRF on RUNX2 incultured periodontal progenitors cells were collected andequal amounts of protein extracts in a lysis buffer containing100mM Tris HCl pH 90 200mM KCl 25mM EGTA36mM MgCl

2 2 deoxycholic acid and 10 DTT vv were

subjected to SDS-polyacrylamide gel electrophoresis Theseparated proteins were transferred to a PVDF membrane(Immobilon P Millipore) and the membrane was incu-bated with anti-RUNX2 or anti-GAPDH primary antibodies(Abcam Cambridge MA USA) Immune complexes weredetected with peroxidase-conjugated secondary antibody(Molecular Probes Carlsbad CA USA) and enhanced bychemiluminescence reagents (Pierce Biotechnology Rock-ford IL USA) The amounts of protein expression werecompared after normalization against GAPDH as an internalcalibrator in each lane

29 Subcutaneous Implant Fresh PRF membranes mea-suring 5mm2 in diameter and 1mm2 in thickness were

implanted under the cutis of nudemice (male 6 weeks of ageCharles River) After 7 and 14 days of implantation nudemicewere sacrificed and implants were dissected for histologicalanalyses

210Mallory Staining Implantswere fixedwith 10 formalinat 4∘C and processed for paraffin sections Deparaffinized andrehydrated sections were stained in a series of toluidine blueacid fuchsine and aniline for 2 minutes respectively

211 Clinical Application Two clinical pilot studies wereconducted to test the effect of PRF on peri-implant sitesin human patients These studies were approved by theEthics Committee of the Jilin University Health ScienceCenter Changchun China Informed consent was obtainedfrom patients before surgery Studies were conducted at JilinUniversity Stomatological Hospital and were consistent withthe principles enunciated in the Declaration of Helsinkion the protection of human subjects All patients selectedfor implant placement and PRF treatment were requiredto demonstrate superior oral hygiene with no or minimalplaque Two Asian healthy adults onemale aged 28 years andone female aged 22 years served as volunteers Both patientsdemonstrated evidence of residual root tips prior to toothextraction according to radiographs For the rehabilitation ofthe patient the fractured incisor (patient I) and the decayedmolar (patient II) were extracted Implant dimensions slightlyvaried between patients (13mm times 35mm patient I and12mm times 48mm patient II) Both patients exhibited sub-stantial gaps between implant and alveolar bone socket Toclose the gap between implant and extraction socket PRFwas prepared individually from each patientrsquos blood sampleand squeezed PRF membranes were placed between implantand adjacent bone Subsequently the surgical site was closedusing silk sutures As a followup radiographs were takenimmediately and three months after surgery Wound healingand bone levels were examined using intraoral photographsand oral radiographs To calculate bone levels implantdimensions served as a reference

212 Statistical Analysis Statistical analysis was performedusing the SPSS 130 software (Chicago IL USA) and Studentrsquos119905-test Data were expressed as mean plusmn SD For all tests statis-tical significance was assigned when 119875 lt 005 Experimentswere repeated 3 times to ensure reproducibility

3 Results

31 PRF Clot Prepared from Fresh Blood Contained FibrinWhite Blood Cells and Leukocytes The focus of the presentstudy was to determine the suitability of PRF for biologicalreengineering of complex periodontal tissues including softand mineralized tissues As a first step we have analyzedthe histological composition of the PRF scaffold used inour studies For our experiments PRF was generated bylow-speed centrifugation from whole blood Centrifugationresulted in separation of three distinct fractions platelet-poorplasma (PPP) platelet-rich fibrin (PRF) and red blood cells


PPP

PRF

RBC

(a) (b) (c)

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WBCand PLT

RBC

(d)

Figure 1 Preparation and structure of platelet-rich fibrin (PRF) (a) shows three fractions of whole blood generated by low-speedcentrifugation platelet-poor plasma (PPP) platelet-rich fibrin (PRF) and red blood cells (RBC) At this stage PRF and RBC have formeda gel while the PPP fraction remains liquid (b) Mechanical separation of the RBC fraction from the PRF fraction after decanting the PPPfraction (c) Generation of solid PRF after liquid removal (d) 5 120583m paraffin section through solidified PRF generated in (c) The sectioncontains three portions (i) the cell-free fibrin clot (ii) the buffy coat portion containing white blood cells (WBC) and platelets (PLT) and(iii) the red blood cell (RBC) portion Note that the majority of the white blood cells and platelets are trapped in the buffy coat

(RBC) (Figure 1(a)) The PRF fraction was further isolatedby decanting the soluble PPP fraction and by mechanicallyremoving the RBC fraction (Figure 1(b)) Liquid removalfrom the PRF fraction through mechanical pressure betweengauze layers resulted in a fairly solid gel-like material (Fig-ure 1(c)) HampE-stained paraffin sections through the PRF clotrevealed three portions (i) the cell-free fibrin clot (ii) thebuffy coat portion containing white blood cells (WBC) andplatelets (PLT) and (iii) the red blood cell (RBC) portion(Figure 1(d)) For the experiments described herein the entirePRF clot was used including fibrin white blood cells andplateletsThe red blood cells immediately attached to the PRFclot were not further separated

32 PRF Significantly Increased Periodontal Progenitor CellMigration and Proliferation In Vitro When Compared to PPPand DMEMMedia Effective biocompatible tissue engineer-ing scaffolds provide a template for cells to migrate andproliferate [20] To determine the ability of PRF-based scaf-folds to promote cell proliferation andmigration periodontalprogenitors were cultured for up to 7 days using PRF PPPor DMEM alone (Figure 2) PRF was compared with PPPto compare the effects of a fibrin-rich (PRF) and a fibrin-poor (PPP) blood centrifugate on periodontal cells Ourcell proliferation assays using PDL fibroblasts dental follicleprogenitors and alveolar bone osteoblasts demonstrated agradual increase in cell density over a culture period of 7days with all three culture conditions (Figures 2(a)ndash2(c))PRF resulted in higher proliferation rates than DMEM

medium alone or platelet-poor plasma (PPP) (Figures 2(a)ndash2(c)) After 7 days of culture the PRF-induced increase inproliferation significantly surpassed the PPP- and DMEM-induced proliferation rate in all cell cultures examined (301for DF 34 for PDL and 224 for AB 119875 lt 005) Migrationassays revealed that PRF caused a highly significant (119875 lt00001) and higher than 15-fold increase (193-fold for DF 18-fold for PDL and 171-fold for AB) in the number of migratedcells when compared to DMEMmedium while platelet-poorplasma (PPP) only caused a moderate 5ndash10-fold increase inthe number of migrated cells (78-fold for DF 5-fold for PDLand 71-fold for AB)

33 PRF Almost Equaled Osteogenic Medium in Its Effect onMineralization Behavior of Cultured Periodontal Progenitorsand in Comparison to DMEM and PPP Previous studieshave established the osteogenic potential of periodontalprogenitors including dental follicle and periodontal liga-ment progenitors [21 22] However periodontal progenitorsdo not form bone or other mineralized tissues in routinetissue engineering applications without osteoinduction [2]Moreover in vivo studies have demonstrated the suitabilityof fibrin-based scaffolds for bone tissue engineering [23] Wetherefore asked the question to what extent PRF unleashesthe osteogenic potential of periodontal progenitors In ourstudies DF PDL and AB cells were subjected to coculturewith platelet-rich fibrin (PRF) osteogenic medium (OM)platelet-poor plasma (PPP) or Dulbeccorsquos modified Eaglersquosmedium (DMEM) and alkaline phosphatase and alizarinred staining were used to evaluate osteoblast activity and


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(d)

Figure 2 Effects of PRF when compared to PPP and medium alone on proliferation and migration of periodontal progenitor populationsFigures 2(a)ndash2(c) illustrate the results ofMTT proliferation assays when dental follicle progenitors (DF (a)) periodontal ligament progenitors(PDL (b)) and alveolar bone osteoblast progenitors (AB (c)) were cultured on PRF-related substrates The three different substrates used inthis proliferation study PRF PPP and DMEMmedia are distinguished by line patterns (Figure 2(a)) (d) Difference in chemotaxis behaviorbetween periodontal progenitors when cultured in PRF-conditioned media PPP and DMEM medium Level of significance was calculatedin comparison to the DMEM-treated cells within each group lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001

mineralized nodule formation After 7 days alkaline phos-phatase levels as a result of PRF coculture increased 41-fold(DF) 37-fold (PDL) and 126-fold (AB) while OM cocultureresulted in an 51-fold (DF) 58-fold (PDL) and 148-fold(AB) increase of alkaline phosphatase levels (Figure 3(a))These effects were highly significant (119875 lt 001) whilethere was no significant difference in alkaline phosphataselevels between PPP-treated cells and DMEM-treated cellsAfter 14 days alkaline phosphatase levels as a result ofPRF coculture increased 7-fold (DF) 108-fold (PDL) and25-fold (AB) while OM coculture resulted in a 106-fold(DF) 157-fold (PDL) and 358-fold (AB) increase of alkalinephosphatase levels (Figure 3(c)) Differences once more werehighly significant (119875 lt 0001 for DFOM 119875 lt 001 forDFPRF 119875 lt 0001 for PDL and 119875 lt 00001 for AB)

while alkaline phosphate levels of PPP-treated and DMEM-treated cells were at the same level Alkaline phosphataselevels after 21 days of culture were decreased compared to14-day levels but still are higher than those after 7 daysSpecifically alkaline phosphatase levels in the PRF-treatedgroup increased 104-fold (DF) 29-fold (PDL) and 201-fold(AB) while alkaline phosphatase levels in the OM-treatedgroup increased 85-fold (DF) 33-fold (PDL) and 223-fold(AB) once more at statistically highly significant levels (119875 lt001 for DF and PDL and 119875 lt 0001 for AB) (Figure 3(e))

In addition to alkaline phosphate levels alizarin redS staining was performed as a means to assess matrixmineralization After 7 days alizarin red S staining as a resultof PRF coculture increased 2-fold (DF) 23-fold (PDL) and49-fold (AB) while OM coculture resulted in an increase


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Figure 3 Effect of PRF PPP and DMEM media on mineralization behavior of periodontal progenitor populations (a) (c) (e) and (g) arealkaline phosphatase staining assays and (b) (d) (f) and (h) are alizarin red S mineralization assays In (a)ndash(f) alkaline phosphatase ((a)(c) and (e)) or alizarin red ((b) (d) and (f)) staining in periodontal progenitor cells cultured for 7 14 and 21 days were compared Differentcoculture conditions (PRF PPP OM and DMEM) are distinguished by different bar patterns which are identified in the bar legend above(b)The three periodontal progenitor populations compared in this study dental follicle (DF) periodontal ligament (PDL) and alveolar bone(AB) are labeled on the 119909-axis of the graphs in (a)ndash(f) (g) and (h) illustrate the differences in mineralization behavior between alveolar boneprogenitors cocultured with PRF OM PPP and DMEM for 14 days (g) is a photograph of the alkaline phosphate-stained 6-well plate and (h)is a photograph of the alizarin red-stained 6-well plate Level of significance was calculated in comparison to the DMEM-treated cells withineach group lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001


of alizarin red S staining by 19-fold (DF) 37-fold (PDL)and 55-fold (AB) (Figure 3(b)) These effects were highlysignificant (119875 lt 005 for DF 119875 lt 001 for PDLOM 119875 lt 005for PDLPRF and 119875 lt 001 for AB) while there was nosignificant difference in alizarin red S staining between PPP-treated cells and DMEM-treated cells After 14 days alizarinred S staining as a result of PRF coculture increased 26-fold(DF) 93-fold (PDL) and 33-fold (AB) while OM cocultureresulted in an increase of alizarin red S staining by 56-fold(DF) 159-fold (PDL) and 52-fold (AB) when comparedto DMEM-treated cells (Figure 3(d)) Differences once morewere highly significant (119875 lt 001 for DFOM 119875 lt 005 forDFPRF 119875 lt 0001 for PDLOM 119875 lt 001 for PDLPRF 119875 lt00001 for ABOM and 119875 lt 0001 for ABPRF) Alizarin redS staining after 21 days of culture was further enhanced withstaining levels in the PRF-treated group increased to 28-fold(DF) 108-fold (PDL) and 202-fold (AB) while alizarin redS staining in the OM-treated group increased 45-fold (DF)14-fold (PDL) and 231-fold (AB) once more at statisticallyhighly significant levels (119875 lt 001 for DF 119875 lt 0001 for PDLand 119875 lt 00001 for AB) (Figure 3(f)) Effects of PRF OMPPP and DMEM on alkaline phosphatase levels (Figure 3(g))and alizarin red S staining (Figure 3(h)) as mineralizationindicators after 14 days of alveolar bone progenitor culturewere documented

34 PRF Significantly Enhanced RUNX2 Expression andReduced MGP Expression in AB Progenitors While It AffectedDF and PDL Cells to a Lesser Degree To test whether changesin mineralization behavior of periodontal progenitors asa result of PRF application or treatment with osteogenicmedium (OM) corresponded to the changes in gene expres-sion levels expression levels of two osteogenic modulatorsthe osteoblastic differentiation transcription factor RUNX2[24] and the extracellular matrix mineralization inhibitor[25] matrix GLA protein (MGP) After 7 days of cultureneither RUNX2 nor MGP was substantially affected by PRFor OM in any of the three cell types investigated (Figures4(a) and 4(b)) After 14 days of culture RUNX2 expressionin all three cell types was significantly increased as a resultof PRF OM and PPP application (Figure 4(c)) Notably PRFand OM treatment had a remarkable effect on RUNX2 levelsin AB cells when compared to DF and PDL cells WhileRUNX2 expression inDF andPDLprogenitors approximatelydoubled in response to OM and PRF exposure RUNX2 levelsincreased 33-fold afterOMapplication and 36-fold after PRFapplication in AB cells (Figure 4(c) 119875 lt 001) This generaltrend was retained after 21 days albeit at slightly reducedlevels OM caused a 18-fold increase in RUNX2 expressionin AB cells and PRF resulted in a 26-fold increase in RUNX2expression in AB cells while there was little effect in the othertwo progenitor cell populations (Figure 4(e)) On westernblots PRF resulted in a 104-fold increase and OM in a 2352-fold increase in RUNX2 levels in AB cells cultured for 2 weekswhen compared to the DMEM group (Figures 4(g) and 4(h)119875 lt 0001) As mentioned above MGP was little affectedby PRF or OM in any of the cell types after one week ofculture (Figure 4(b)) Two or three weeks in the presence ofPPP resulted in an approximately 2-fold elevation ofMGP in

all three cell types (Figures 4(d) and 4(f) 119875 lt 005) Mostnotable was a 31-fold reduction inMGP expression as a resultof PRF application in AB cells after 2 weeks (119875 lt 001) and a2-fold PRF-induced reduction inMGP expression in DF cells(Figures 4(d) and 4(f) 119875 lt 005) There was still a 12-foldreduction inMGP expression in AB progenitors as a result ofPRF treatment after 3 weeks of culture while there were fewdifferences in all other groups and cells after three weeks ofculture with the exception of the MGP upregulation causedby PPP (Figure 4(f))

35 Fibrin-Enhanced Alkaline Phosphatase Activity and Min-eralization in AB Progenitors Studies so far indicated thatPRF affects mineralization of periodontal progenitors espe-cially AB progenitors To determine whether fibrin as themajor structural component of the PRF scaffold affects min-eralization behavior of periodontal progenitors cells werecultured on fibrin-coated culture dishes vacuum gas plasma-treated tissue culture dishes and untreated Petri dishesfor 5 days and stained for alkaline phosphatase activity(Figure 5(a)) or alizarin red S (Figure 5(b)) Alkaline phos-phatase levels of PDL and AB progenitors were significantlyelevated on fibrin-coated dishes when compared to vacuumgas plasma-coated dishes (457-fold for AB and 39-fold forPDL) while there was only a 177 elevation of DF cell pro-genitor alkaline phosphatase levels on fibrin-coated dishesIn contrast alizarin red S as an indicator of mineralizationwas significantly increased in the AB progenitor group whencompared to the PDL (476-fold increase when comparingAB versus PDL) and the DF groups (326-fold increasewhen comparing AB versus DF) AB culture alizarin red Slevels were 215-fold higher on fibrin-coated dishes than oncommercially pretreated dishes

36 PRF Scaffolds Integrated with Surrounding Tissues andWere Partially Replaced with Collagen Fibers Two Weeksafter Subcutaneous Implantation in NudeMice To determinethe suitability of PRF as a scaffold for tissue regenera-tion PRF membranes were subcutaneously implanted innude mice One week after implantation PRF membraneswere completely surrounded by subcutaneous collagen fibers(Figure 6(a)) After 14 days the thickness of the membranewas reduced to 05mm and the remaining tissue had beenreplaced by dense collagen fibers (Figures 6(b) and 6(d)) Atthis time point pores inside of the PRFmembrane containedmeshworks of thin collagen fibrils (Figure 6(c))

37 Soft Tissue Healing and New Bone Formation after PRFApplication for Peri-Implant Periodontal Regeneration Totest the effect of PRF on peri-implant sites in human patientsclinical pilot studies were conducted In these studies PRFwas placed into peri-implant tissue gaps of both humanmolars and incisors (Figure 7) Intraoral images illustratesoft tissue healing 5 days after surgery and PRF membraneplacement (Figure 7(h)) as well as complete integration threemonths after surgery (Figures 7(c) and 7(i)) Radiographsdemonstrate substantial new alveolar bone formation sur-rounding both implants (Figures 7(e) and 7(l) versus Figures7(d) and 7(k)) On the mesial aspect of the incisor implant


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6

5

4

3

2

1

0

Relat

ive e

xpre

ssio

n

DF PDL AB

MGP 21d

(f)

62

37

PRF OM PPP DMEM

RUNX2

GAPDH

(g)

600

480

360

240

120

0

Inte

grat

ed d

ensit

y

PRF OM PPP DMEM

lowastlowastlowast

lowastlowastlowast

(h)

Figure 4 Differences in mineralization-associated gene expression patterns in cocultures of periodontal progenitors and centrifuged bloodderivatives (a) (c) and (e) are real-time RT-PCR assays for the osteoblast transcription factor Runx2 and (b) (c) and (f) are real-time RT-PCR assays for the calcification inhibitorMatrix Gla Protein (MGP) Different coculture conditions (PRF PPP andDMEM) are distinguishedby different bar patterns identified in the upper right corner of the figureThe three periodontal progenitor populations compared in this studydental follicle (DF) periodontal ligament (PDL) and alveolar bone (AB) are labeled on the 119909-axis of the graphs in (a)ndash(f) (g) is a westernblot comparing Runx2 protein levels generated by AB cells cocultured either with PRF OM PPP or DMEM GAPDH served as a control(h) is the corresponding densitometry evaluation Level of significance was calculated in comparison to the DMEM-treated cells within eachgroup lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001


DF PDL AB

Alkaline phosphatase

Vacuum gasplasma treated

Fibrin coated

Untreated

(a)


Alizarin red S

DF PDL AB

Fibrin coated

Untreated

(b)

Figure 5 Effect of the major PRF component fibrin on periodontal progenitor attachment and mineralization Osteogenic lineagedifferentiation (a) and mineralized nodule formation (b) of dental follicle cells (DF) periodontal progenitors (PDL) and alveolar boneprogenitors (AB) on fibrin-coated dishes vacuum gas plasma-treated tissue culture dishes and untreated Petri dishes as revealed by alkalinephosphatase (a) and alizarin red S (b) Alkaline phosphatase activity was determined after one week and alizarin red S staining was performedafter three weeks

7d

PRF

200120583m

(a)

PRF

14d

Coll

(b)

PRF

14d

50120583m

lowast

(c)

14d

Coll

(d)

Figure 6 Evaluation of PRF as a scaffold in nude mice subcutaneous implantsThis figure contains micrographs of paraffin sections throughthe center of the implant stained using Malloryrsquos connective tissue stain (a) After 7 days of implantation the PRF implant remained fairlyintact andwas only surrounded by collagen fibers in the periphery of the implant (b) After 14 days of implantation the size of the PRF scaffold(PRF) was reduced and mostly replaced with collagen fibers (Coll here stained in blue) (c) and (d) illustrate histology of the 14-day implantat higher magnification Note small collagen fibers (light blue) present within the pores of the implant (lowast (c)) (d) demonstrates new collagenfiber formation (Coll) replacing the PRF implant (a) and (b) bar = 200 120583m (c) and (d) bar = 50 120583m


25 cm

(a)

PRF

(b)

(c) (d) (e)

(f) (g)

PRF

(h) (i)

(j) (k) (l)

Figure 7 PRF application simultaneous with dental implant surgery (a)ndash(e) Case I and (f)ndash(l) Case II (a)ndash(c) and (f)ndash(i) are intraoralmicrographs and (d) (e) and (j)ndash(l) are X-rays Case I (a) reveals a 25mm gap at the mesial aspect of the implant replacing the upperleft incisor (b) illustrates PRF placement in the gap between implant and adjacent alveolar bone (c) demonstrates healing of the implantsite and healthy gingiva three months after implant placement (d) X-ray documenting gap between implant and alveolar bone immediatelyafter implant placement (Case I) and (e) X-ray demonstrating new alveolar bone formation at the site of PRF application three months afterimplant placement (Case I) Case II (f) extraction socket of a lower left molar immediately after tooth extraction (g) implant placementand filling of bone-deficient peri-implant space with PRF (h) healing of surgery site and PRF implant five days after surgery (i) healthy softtissue surrounding implant three months after implant placement (j) X-ray of decayed lower left molar treated in Case II prior to extraction(k) X-ray of implant taken immediately after surgery (l) X-ray illustrating new alveolar bone formation three months after surgery Note thedisappearance of the bony defect between implant and adjacent sockets The black bar drawn in the implant center on the radiographs ((d)(e) (k) and (l)) represents the distance from the foramen apical to the cervical-most margin of the surrounding alveolar bone


there was a 54mm alveolar bone gain (Figure 7(e) versusFigure 7(d)) while there was bone gain on both aspectsof the molar implant amounting to approximately 49mm(Figure 7(l) versus Figure 7(k)) Newly formed alveolar bonewas radioopaque and contained trabeculae (Figures 7(e) and7(l))

4 Discussion

In the present study a series of experiments was conductedto determine the usefulness of PRF as a bioactive scaffold forperiodontal tissue regeneration In a first set of experimentsthe effect of PRF on proliferation and migration of DFPDL and AB progenitors was investigated DF PDL and ABprogenitors were chosen as metabolically active periodontalprogenitor populations [1] In a second set of studies theeffect of PRF on periodontal progenitor mineralization wasdetermined in vitro In these experiments classic osteoblastdifferentiation and mineralization markers such as alkalinephosphatase and alizarin red S as well as RUNX2 expressionwere employed to gain detailed insight into the effect of PRFonmineralization behavior of periodontal progenitors To askwhether themajor PRF component fibrinwas associatedwiththe effect of PRF on periodontal progenitor mineralizationour alkaline phosphatase and alizarin red S assays wereconducted on fibrin-coated dishes Finally subcutaneousimplantation studies and human pilot studies were per-formed to determine the applicability of PRF as a scaffoldfor periodontal tissue regeneration Together these studiesdemonstrated that the application of PRF in periodontalregeneration had twomajor benefits (i) the promotion of softtissue healing as explained by the effect of PRF on progenitorproliferation and migration and (ii) the induction of newalveolar bone formation as possibly facilitated by the fibrin-mediated effect of PRF on RUNX2 expression osteoblastdifferentiation and matrix mineralization and also by thealkaline phosphatase activity-stimulating effect of fibrin

The PRFmembranes used for the present study containedthe entire PRF clot centrifuged from fresh blood includingfibrin leukocytes and platelets By the very nature of thispreparation the PRF membranes used for the current studywere three-component scaffolds that were not further bio-chemically dissected for individual components as the bio-logical and therapeutic effects of the PRF scaffold have beenreported to depend on the fresh preparation of an autologousblood fraction [4] Moreover the compound PRF membranecontains not only the structural scaffold components fibrinleukocytes and platelets but also a multitude of growthfactors such as TGF-1205731 VEGF IGFs and PDGF-AB as wellas matricellular proteins such as thrombospondin-1 [26] Inthe present study the composition of the PRFmembrane waskept as homogeneous as possible by maintaining strict andreproducible preparation conditionsWe expect some limitedvariability among PRF samples due to differences betweenblood of individual host organisms including humansNevertheless our studies reported excellent reproducibil-ity between individual PRF preparations from differentdonor individuals in terms of mineralization proliferationand migration effects indicating that for the purpose of

the present study our PRFmembranes elicited highly repeat-able biological effects

Our data demonstrated that PRF significantly improvedperiodontal progenitor cell proliferation and migration invitro when compared to PPP and DMEM media The effectof PRF on cell proliferation is well established and has beendescribed in awide variety of cell types including periodontalligament cells osteoblasts gingival fibroblasts oral epithelialcells BMSCs preadipocytes and prekeratinocytes [9 27]PRFrsquos role in the stimulation of cell proliferation may be dueto a gradual release of growth factors [8] some of whichmight be released by platelets and others might have beentrapped within the fibrin scaffold and gradually diffusedinto the culture medium Even though both PRF and PPPmedia are blood plasma preparations PRF had a significantlystronger effect on proliferation than PPP We attribute thiseffect to different subsets of cytokines in PRF and PPP andto the controlled release of cytokines trapped in PRF fibrinmeshes [28] Interestingly there was a stronger effect of PRFon AB and DF cells than on PDL progenitors and the PRF-induced elevation of proliferation occurred earlier in ABprogenitors than in the other two cell types investigatedsuggesting that the effects of PRF are tissue specific and favorAB osteoblasts over the other two cell types studied hereWhile the PRF-induced enhancement of cell proliferationremained in the 30 range over PPP or DMEM controls theeffect of PRF on cell migration was much more pronouncedfeaturing a 25-fold elevation compared to PPP and anapproximately 20-fold elevation when compared to DMEMThis dramatic PRF-induced increase in cell migration hasnot been reported previously and might be explained bychemokines released by leukocytes trapped in PRF [29] or bythe effect of soluble fibrin components on cell migration [30]

From a clinical perspective the reported effects of PRF onbone regeneration [31 32] are of great interest for orthopedicapplications because of the limitations of current strategiesto enhance bone regeneration [33] Moreover alveolar ridgeaugmentation is of great benefit to implant dentistry becauseof the lack of supporting bone for implant placement [34]Our in vitro studies indicated that PRF almost equaledosteogenic medium in its effect on mineralization behaviorof cultured periodontal progenitors surpassing DMEM andPPP by a significant margin While the effect of PRF onosteoblast alkaline phosphatase levels [8] and on the for-mation of mineral nodules [35] has already been reportedwe have demonstrated here that PRF significantly enhancesthe expression of the osteoblast differentiation transcriptionfactor RUNX2 and reduces expression of the mineralizationinhibitor MGP preferentially in alveolar bone osteoblastprogenitors and less so in dental follicle cells and periodontalligament progenitors We propose that this tissue-specificenhancement of osteoblastmineralizationmight be due to theenhanced alkaline phosphatase activity induced by the fibrincomponent of PRF (shown in the present study) and to thegreater susceptibility of osteoblast cells to differentiate alongosteogenic lineage pathways

Both our in vitro and our clinical studies indicate thatthe benefit of PRF for bone regenerative procedures lies in itscombined competency as a cell proliferation migration and


wound-healing agent together with its tissue-specific abilityto promote osteoblast differentiation and new bone forma-tion Our clinical pilot experiments revealed peri-implantbone gain of approximately 5mm in conjunction with softtissue healing around the implant sitemdasha highly desirableoutcome in which we attribute the combined biologicaleffects of PRF on gingival fibroblast and periodontal ligamentcellmigration aswell as alveolar bone osteoblast proliferationand mineralization Moreover PRF is a biodegradable scaf-fold as our studies have demonstrated that PRF subcutaneousimplants were readily replaced with dense collagen evenafter 2 weeks of implant placement in nude mice suggestingexcellent biodegradability The combinatorial effects of PRFon soft tissue healing and bone regeneration may limit its usein regenerativemedicine applications in which calcification isnot a desired outcome however our studies indicate that PRFcontains a number of attributes ideally suited for its use as ascaffold for alveolar ridge augmentation and bone healing

Conflict of Interests

The authors declare no conflict of interests

Acknowledgment

The authors gratefully acknowledge the Research OpenAccess Publishing (ROAAP) Fund of theUniversity of Illinoisat Chicago for financial support towards the open accesspublishing fee for this paper

References

[1] S J Dangaria Y Ito CWalker R Druzinsky X Luan and T GH Diekwisch ldquoExtracellularmatrix-mediated differentiation ofperiodontal progenitor cellsrdquoDifferentiation vol 78 no 2-3 pp79ndash90 2009

[2] S J Dangaria Y Ito X Luan and T G Diekwisch ldquoSuccessfulperiodontal ligament regeneration by periodontal progenitorpreseeding on natural tooth root surfacesrdquo Stem Cells vol 20no 10 pp 1659ndash1668 2011

[3] A Hollander P Macchiarini B Gordijn and M BirchallldquoThe first stem cell-based tissue-engineered organ replacementimplications for regenerative medicine and societyrdquo Regenera-tive Medicine vol 4 no 2 pp 147ndash148 2009

[4] J Choukroun F Adda C Schoeffer and A Vervelle ldquoPRF anopportunity in perio- implantologyrdquo Implantodontie vol 42 pp55ndash62 2000

[5] D M Dohan J Choukroun A Diss et al ldquoPlatelet-richfibrin (PRF) a second-generation platelet concentrate Part IIplatelet-related biologic featuresrdquo Oral Surgery Oral MedicineOral Pathology Oral Radiology and Endodontology vol 101 no3 pp E45ndashE50 2006

[6] W Bensaıd J T Triffitt C Blanchat K Oudina L Sedel andHPetite ldquoA biodegradable fibrin scaffold for mesenchymal stemcell transplantationrdquoBiomaterials vol 24 no 14 pp 2497ndash25022003

[7] Y H Kang S H Jeon J Y Park et al ldquoPlatelet-rich fibrinis a bioscaffold and reservoir of growth factors for tissueregenerationrdquoTissue Engineering A vol 17 no 3-4 pp 349ndash3592011

[8] L He Y Lin X Hu Y Zhang andHWu ldquoA comparative studyof platelet-rich fibrin (PRF) and platelet-rich plasma (PRP) onthe effect of proliferation and differentiation of rat osteoblastsin vitrordquo Oral Surgery Oral Medicine Oral Pathology OralRadiology and Endodontology vol 108 no 5 pp 707ndash713 2009

[9] D M D Ehrenfest A Diss G Odin P Doglioli M PHippolyte and J B Charrier ldquoIn vitro effects of ChoukrounrsquosPRF (platelet-rich fibrin) on human gingival fibroblasts dermalprekeratinocytes preadipocytes and maxillofacial osteoblastsin primary culturesrdquo Oral Surgery Oral Medicine Oral Pathol-ogy Oral Radiology and Endodontology vol 108 no 3 pp 341ndash352 2009

[10] J Choukroun A Diss A Simonpieri et al ldquoPlatelet-richfibrin (PRF) a second-generation platelet concentrate Part IVclinical effects on tissue healingrdquo Oral Surgery Oral MedicineOral Pathology Oral Radiology and Endodontology vol 101 no3 pp E56ndashE60 2006

[11] M T Peck J Marnewick and L Stephen ldquoAlveolar ridgepreservation using leukocyte and platelet-rich fibrin a report ofa caserdquo Case Reports in Dentistry vol 2011 Article ID 3450485 pages 2011

[12] A Sharma and A R Pradeep ldquoTreatment of 3-wall intrabonydefects in patients with chronic periodontitis with autologousplatelet-rich fibrin a randomized controlled clinical trialrdquoJournal of Periodontology vol 82 no 12 pp 1705ndash1712 2011

[13] M Thorat A R Pradeep and B Pallavi ldquoClinical effectof autologous platelet-rich fibrin in the treatment of intra-bony defects a controlled clinical trialrdquo Journal of ClinicalPeriodontology vol 38 no 10 pp 925ndash932 2011

[14] F Inchingolo M Tatullo M Marrelli et al ldquoTrial with platelet-rich fibrin and Bio-Oss used as grafting materials in thetreatment of the severe maxillar bone atrophy clinical andradiological evaluationsrdquo European Review for Medical andPharmacological Sciences vol 14 no 12 pp 1075ndash1084 2010

[15] Y Zhang S Tangl C D Huber Y Lin L Qiu and XRausch-Fan ldquoEffects of Choukrounrsquos platelet-rich fibrin onbone regeneration in combination with deproteinized bovinebone mineral in maxillary sinus augmentation a histologicaland histomorphometric studyrdquo Journal of Cranio-MaxillofacialSurgery vol 40 no 4 pp 321ndash328 2011

[16] C H Tsai S Y Shen J H Zhao and Y C Chang ldquoPlatelet-rich fibrin modulates cell proliferation of human periodontallyrelated cells in vitrordquo Journal of Dental Sciences vol 4 no 3 pp130ndash135 2009

[17] Y C Chang and J H Zhao ldquoEffects of platelet-rich fibrinon human periodontal ligament fibroblasts and application forperiodontal infra-bony defectsrdquo Australian Dental Journal vol56 no 4 pp 365ndash371 2011

[18] D M Dohan J Choukroun A Diss et al ldquoPlatelet-richfibrin (PRF) a second-generation platelet concentrate PartI technological concepts and evolutionrdquo Oral Surgery OralMedicine Oral Pathology Oral Radiology and Endodontologyvol 101 no 3 pp E37ndashE44 2006

[19] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the2minusΔΔ119862T methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[20] E Sachlos and J T Czernuszka ldquoMaking tissue engineeringscaffolds work Review the application of solid freeform fab-rication technology to the production of tissue engineeringscaffoldsrdquo European Cells amp Materials Journal vol 5 pp 29ndash392003


[21] X Luan Y Ito S Dangaria and T G H Diekwisch ldquoDentalfollicle progenitor cell heterogeneity in the developing mouseperiodontiumrdquo Stem Cells and Development vol 15 no 4 pp595ndash608 2006

[22] S Tsuchiya S Ohshima Y Yamakoshi J P Simmer and M JHonda ldquoOsteogenic differentiation capacity of porcine dentalfollicle progenitor cellsrdquo Connective Tissue Research vol 51 no3 pp 197ndash207 2010

[23] J M Karp F Sarraf M S Shoichet and J E Davies ldquoFibrin-filled scaffolds for bone-tissue engineering an in vivo studyrdquoJournal of Biomedical Materials Research A vol 71 no 1 pp162ndash171 2004

[24] R T Franceschi and G Xiao ldquoRegulation of the osteoblast-specific transcription factor Runx2 responsiveness to multiplesignal transduction pathwaysrdquo Journal of Cellular Biochemistryvol 88 no 3 pp 446ndash454 2003

[25] B Newman L I Gigout L SudreM E Grant andG AWallisldquoCoordinated expression of matrix Gla protein is requiredduring endochondral ossification for chondrocyte survivalrdquoJournal of Cell Biology vol 154 no 3 pp 659ndash666 2001

[26] D M D Ehrenfest G M de Peppo P Doglioli and G Sam-martino ldquoSlow release of growth factors and thrombospondin-1 in Choukrounrsquos platelet-rich fibrin (PRF) a gold standardto achieve for all surgical platelet concentrates technologiesrdquoGrowth Factors vol 27 no 1 pp 63ndash69 2009

[27] Q Li Y Geng L Lu T Yang M Zhang and Y ZhouldquoPlatelet-rich fibrin-induced bone marrow mesenchymal stemcell differentiation into osteoblast-like cells and neural cellsrdquoNeural Regeneration Research vol 6 no 31 pp 2419ndash2423 2011

[28] F Clipet S Tricot N Alno et al ldquoIn vitro effects of Choukrounrsquosplatelet-rich fibrin conditioned medium on 3 different cell linesimplicated in dental implantologyrdquo Implant Dentistry vol 21no 1 pp 51ndash56 2012

[29] D M Dohan J Choukroun A Diss et al ldquoPlatelet-richfibrin (PRF) a second-generation platelet concentrate Part IIIleucocyte activation a new feature for platelet concentratesrdquoOral Surgery OralMedicine Oral Pathology Oral Radiology andEndodontology vol 101 no 3 pp E51ndashE55 2006

[30] R A F Clark J M Lanigan and P DellaPelle ldquoFibronectin andfibrin provide a provisional matrix for epidermal cell migrationduring wound reepithelializationrdquo Journal of Investigative Der-matology vol 79 no 5 pp 264ndash269 1982

[31] J Choukroun A Diss A Simonpieri et al ldquoPlatelet-rich fibrin(PRF) a second-generation platelet concentrate Part V histo-logic evaluations of PRF effects on bone allograft maturationin sinus liftrdquo Oral Surgery Oral Medicine Oral Pathology OralRadiology and Endodontology vol 101 no 3 pp 299ndash303 2006

[32] A Simonpieri J Choukroun M D Corso G Sammartino andD M D Ehrenfest ldquoSimultaneous sinus-lift and implantationusing microthreaded implants and leukocyte- and platelet-richfibrin as sole grafting material a six-year experiencerdquo ImplantDentistry vol 20 no 1 pp 2ndash12 2011

[33] R Dimitriou E Jones D McGonagle and P V GiannoudisldquoBone regeneration current concepts and future directionsrdquoBMCMedicine vol 3 no 9 p 66 2011

[34] F Carinci A Farina U Zanetti et al ldquoAlveolar ridge augmen-tation a comparative longitudinal study between calvaria andiliac crest bone grafrsrdquoThe Journal of oral implantology vol 31no 1 pp 39ndash45 2005

[35] D M D Ehrenfest P Doglioli G M de Peppo M Del Corsoand J B Charrier ldquoChoukrounrsquos platelet-rich fibrin (PRF)

stimulates in vitro proliferation and differentiation of humanoral bone mesenchymal stem cell in a dose-dependent wayrdquoArchives of Oral Biology vol 55 no 3 pp 185ndash194 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


illustrated in a number of studies revealing proliferation anddifferentiation of osteoblasts and gingival fibroblasts [8 9]Clinical studies have demonstrated that PRF promotes softtissue and bone regeneration [10 11] as well as periodontaltissue regeneration [12 13]The ability of PRF to augment andregenerate compromised tissues may be enhanced in combi-nation with bone substitutes such as Bio-Oss or autologousbone [14 15] Together these studies have established PRF as ahighly biocompatible and inductive scaffold useful for a broadrange of tissue engineering applications

In the present study we have hypothesized that PRF mayprovide a scaffold material for periodontal tissue regener-ation Earlier studies have reported the effect of PRF onperiodontal cells [16 17] Here we have compared the effectsof PRF on dental follicle periodontal ligament and alveolarbone cells [1] using microscopy as well as biological assaysfor migration proliferation mineralization and gene expres-sion We also tested PRF scaffold properties in subcutaneousimplants in nude mice Finally we report on the use of PRFas a scaffold for peri-implant alveolar bone augmentationin clinical use Together these studies for the first timecharacterize the tissue-specific biological effects of PRF as abioactive scaffold to promote periodontal soft and hard tissueregeneration

2 Materials and Methods

21 Preparation of PRF To prepare PRF 10mL of freshblood from the precaval vein of a pig was collected into10mL glass-coated tubes without anticoagulants All pigsused in this study were female with an average age of 31months (range from 29 to 35 months) The platelet countof whole pig blood was 105120583L This study was approved bythe Ethics Committee of the University of Illinois at ChicagoUSA As described in previous studies [4 18] samples wereimmediately centrifuged at 2100 rpm (approximately 400 g)for 12 minutes using a Beckman centrifuge The PRF clotswere concentrated between the red blood cell corpusclesat the bottom of the centrifuge tubes and acellular plasmacalled platelet-poor plasma (PPP) at the top of the tubesPPP was collected by pipetting the supernatant of the cen-trifuged blood preparation After PPP removal PRF clotswere mechanically separated from red blood cells and gentlycompressed using gauze to drain the remaining fluid

Reproducibility of studies was ensured by maintain-ing consistent preparation conditions for PRF membranesincluding centrifugation force and time tube size and plateletcount of pig blood and by completing blood collectionprocedures within 2 minutes For in vitro studies all cellculture wells were treated with one whole PRF clot consistingof identical amounts of fibrin platelets and white blood cellsand each PRF clot was freshly prepared from blood

22 Preparation of Conditioned Medium To prepare condi-tioned medium PRF membranes were soaked in 5mL freshDMEM medium without fetal bovine serum in 6-well cellculture plates The conditioned medium was collected every48 h and fresh medium was added into wells after collection

23 Isolation of Human Dental Progenitor Cells To generatehuman dental progenitor cells healthy human teeth (patientsranging from 12 to 15 years) were extracted for orthodonticreasons in accordance with the human subjects protocolapproved byUICrsquos Institutional Review Boards and theOfficefor the Protection Research Subjects The dental follicle (DF)was dissected from developing tooth organs and alveolarbone (AB) and periodontal ligament (PDL) were preparedfrom teeth with tooth roots already formed Mesenchymalstem cells were isolated from the dental tissues after digestionwith collagenasedispase as described before [2]

24 MTT Cell Proliferation Assay PDL DF and AB cells(104 cellswell) were seeded into 96-well plates and culturedfor 8 hours After cells were attached each well was washedtwice with phosphate-buffered saline solution (PBS) andeither PRF-conditionedmedium + 10 FBS or DMEM+ 10PPP + 10 FBS was added to the well DMEM + 10 FBSwas used as a control Prior to the termination of culture cellswere incubated inMTT solution (2120583gmL ofMTT inDMEMwith 2 FBS) for 4 hours To quantify proliferative activitythe MTT-stained cells were lysed in HCLisopropanol andthe absorbance was detected at 570 nm with backgroundsubtraction at 630 nm Cell proliferation was detected on adaily basis over the entire 7-day culture period

25 Chemotaxis Assay Cellmigration assayswere performedusing a FluoroBlok-24-multiwell insert system (DB Bio-sciences Bedford MA) PRF-conditioned medium DMEM+ 10 PPP or DMEM was added into separate wells of thelower chambers of the FluoroBlok system Serum-starvedcells (105) were seeded into each insert and allowed tomigrateto the bottomof themembrane for 12 hoursThenonmigratedcells on the upper surface of the membrane were removedwith a cotton swab and themigratory cells that were attachedto the lower surface were stained with DAPI The numbersof migrated cells per membrane were counted under a LeicaDMRX fluorescent microscope

26 Induction of Osteogenic Differentiation Alkaline Phos-phatase (ALP) Activity Assessment and Alizarin Red S Stain-ing PDL DF and AB were seeded into 6-well plates at aconcentration of 104 cellswell and cultured for 8 hours Aftercell attachment PRF membranes were added to individualwells soaked and subjected to osteogenic treatment whichincluded addition of osteogenic medium (OM) DMEM +10 PPP + 10 FBS or DMEM to each individual wellThe following four groups were employed in this study(i) PRF in DMEM with 10 FBS (ii) 10 PPP in DMEMwith 10 FBS (iii) osteogenic medium with 10 FBS and(iv) DMEM with 10 FBS To test the effect of fibrinas the major PRF component on periodontal progenitordifferentiation and mineralization fibrin was used to coatotherwise untreated cell culture dishes and compared withvacuum gas plasma-treated and plasma-untreated dishes Togenerate fibrin equal amounts of fibrinogen (100mgmL)and thrombin (500 120583mL) were mixed prior to applicationSubsequently treated cells were cultured for an additional 7












3 Results



PPP

PRF

RBC

(a) (b) (c)

Fibrinclot

WBCand PLT

RBC

(d)







16

12

08

04

00

Opt

ical

den

sity

DF

1 2 3 4 5 6 7

Days of culture

PRFPPPDMEM

(a)

PRFPPPDMEM

12

08

04

00

Opt

ical

den

sity

PDL

1 2 3 4 5 6 7

Days of culture

(b)

12

08

04

00

Opt

ical

den

sity

AB

1 2 3 4 5 6 7

Days of culture

PRFPPPDMEM

(c)

25

20

15

10

5

0

Rel

num

ber o

f mig

rate

d ce

lls

DF PDL AB

PRFPPPDMEM



lowastlowast

(d)






30

25

20

15

10

5

0

Inte

grat

ed o

ptic

al d

ensit

y

DF PDL AB

lowast

lowast

ALP 7d

(a)

50

40

30

20

10

0

Inte

grat

ed o

ptic

al d

ensit

y

DF PDL AB

Min 7d

(b)

240

200

160

120

80

40

0

Inte

grat

ed o

ptic

al d

ensit

y

DF PDL AB

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

ALP 14d

(c)

75

60

45

30

15

0In

tegr

ated

opt

ical

den

sity

DF PDL AB

lowast lowast

lowastlowast


lowastlowastlowast


(d)

60

50

40

30

20

10

0

Inte

grat

ed o

ptic

al d

ensit

y


lowastlowastlowast

DF PDL AB

ALP 21d


PRFPPP

(e)


PRFPPP

100

80

60

40

20

0

Inte

grat

ed o

ptic

al d

ensit

y


lowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

DF PDL AB

Min 21d

(f)

PRF

PPP

OM

DMEM

(g)

PRF

PPP

OM

DMEM

(h)










6

5

4

3

2

1

0

Rela

tive e

xpre

ssio

n

DF PDL AB

RUNX2 7d

(a)

6

5

4

3

2

1

0

Relat

ive e

xpre

ssio

n

DF PDL AB

MGP 7d

(b)

6

5

4

3

2

1

0

Relat

ive e

xpre

ssio

n

DF PDL AB

RUNX2 14d



lowast

(c)

6

5

4

3

2

1

0

Relat

ive e

xpre

ssio

n

DF PDL AB

MGP 14d

lowastlowast

lowastlowastlowast

lowast

(d)

0

6

5

4

3

2

1

Relat

ive e

xpre

ssio

n

DF PDL AB

RUNX2 21d

lowastlowast

lowast


PRFPPP

(e)


PRFPPP

6

5

4

3

2

1

0

Relat

ive e

xpre

ssio

n

DF PDL AB

MGP 21d

(f)

62

37

PRF OM PPP DMEM

RUNX2

GAPDH

(g)

600

480

360

240

120

0

Inte

grat

ed d

ensit

y

PRF OM PPP DMEM

lowastlowastlowast

lowastlowastlowast

(h)



DF PDL AB



Fibrin coated

Untreated

(a)


Alizarin red S

DF PDL AB

Fibrin coated

Untreated

(b)


7d

PRF

200120583m

(a)

PRF

14d

Coll

(b)

PRF

14d

50120583m

lowast

(c)

14d

Coll

(d)



25 cm

(a)

PRF

(b)

(c) (d) (e)

(f) (g)

PRF

(h) (i)

(j) (k) (l)




4 Discussion











Acknowledgment


References













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












3 Results



PPP

PRF

RBC

(a) (b) (c)

Fibrinclot

WBCand PLT

RBC

(d)







16

12

08

04

00

Opt

ical

den

sity

DF

1 2 3 4 5 6 7

Days of culture

PRFPPPDMEM

(a)

PRFPPPDMEM

12

08

04

00

Opt

ical

den

sity

PDL

1 2 3 4 5 6 7

Days of culture

(b)

12

08

04

00

Opt

ical

den

sity

AB

1 2 3 4 5 6 7

Days of culture

PRFPPPDMEM

(c)

25

20

15

10

5

0

Rel

num

ber o

f mig

rate

d ce

lls

DF PDL AB

PRFPPPDMEM



lowastlowast

(d)






30

25

20

15

10

5

0

Inte

grat

ed o

ptic

al d

ensit

y

DF PDL AB

lowast

lowast

ALP 7d

(a)

50

40

30

20

10

0

Inte

grat

ed o

ptic

al d

ensit

y

DF PDL AB

Min 7d

(b)

240

200

160

120

80

40

0

Inte

grat

ed o

ptic

al d

ensit

y

DF PDL AB

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

ALP 14d

(c)

75

60

45

30

15

0In

tegr

ated

opt

ical

den

sity

DF PDL AB

lowast lowast

lowastlowast


lowastlowastlowast


(d)

60

50

40

30

20

10

0

Inte

grat

ed o

ptic

al d

ensit

y


lowastlowastlowast

DF PDL AB

ALP 21d


PRFPPP

(e)


PRFPPP

100

80

60

40

20

0

Inte

grat

ed o

ptic

al d

ensit

y


lowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

DF PDL AB

Min 21d

(f)

PRF

PPP

OM

DMEM

(g)

PRF

PPP

OM

DMEM

(h)










6

5

4

3

2

1

0

Rela

tive e

xpre

ssio

n

DF PDL AB

RUNX2 7d

(a)

6

5

4

3

2

1

0

Relat

ive e

xpre

ssio

n

DF PDL AB

MGP 7d

(b)

6

5

4

3

2

1

0

Relat

ive e

xpre

ssio

n

DF PDL AB

RUNX2 14d



lowast

(c)

6

5

4

3

2

1

0

Relat

ive e

xpre

ssio

n

DF PDL AB

MGP 14d

lowastlowast

lowastlowastlowast

lowast

(d)

0

6

5

4

3

2

1

Relat

ive e

xpre

ssio

n

DF PDL AB

RUNX2 21d

lowastlowast

lowast


PRFPPP

(e)


PRFPPP

6

5

4

3

2

1

0

Relat

ive e

xpre

ssio

n

DF PDL AB

MGP 21d

(f)

62

37

PRF OM PPP DMEM

RUNX2

GAPDH

(g)

600

480

360

240

120

0

Inte

grat

ed d

ensit

y

PRF OM PPP DMEM

lowastlowastlowast

lowastlowastlowast

(h)



DF PDL AB



Fibrin coated

Untreated

(a)


Alizarin red S

DF PDL AB

Fibrin coated

Untreated

(b)


7d

PRF

200120583m

(a)

PRF

14d

Coll

(b)

PRF

14d

50120583m

lowast

(c)

14d

Coll

(d)



25 cm

(a)

PRF

(b)

(c) (d) (e)

(f) (g)

PRF

(h) (i)

(j) (k) (l)




4 Discussion











Acknowledgment


References













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


PPP

PRF

RBC

(a) (b) (c)

Fibrinclot

WBCand PLT

RBC

(d)







16

12

08

04

00

Opt

ical

den

sity

DF

1 2 3 4 5 6 7

Days of culture

PRFPPPDMEM

(a)

PRFPPPDMEM

12

08

04

00

Opt

ical

den

sity

PDL

1 2 3 4 5 6 7

Days of culture

(b)

12

08

04

00

Opt

ical

den

sity

AB

1 2 3 4 5 6 7

Days of culture

PRFPPPDMEM

(c)

25

20

15

10

5

0

Rel

num

ber o

f mig

rate

d ce

lls

DF PDL AB

PRFPPPDMEM



lowastlowast

(d)






30

25

20

15

10

5

0

Inte

grat

ed o

ptic

al d

ensit

y

DF PDL AB

lowast

lowast

ALP 7d

(a)

50

40

30

20

10

0

Inte

grat

ed o

ptic

al d

ensit

y

DF PDL AB

Min 7d

(b)

240

200

160

120

80

40

0

Inte

grat

ed o

ptic

al d

ensit

y

DF PDL AB

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

ALP 14d

(c)

75

60

45

30

15

0In

tegr

ated

opt

ical

den

sity

DF PDL AB

lowast lowast

lowastlowast


lowastlowastlowast


(d)

60

50

40

30

20

10

0

Inte

grat

ed o

ptic

al d

ensit

y


lowastlowastlowast

DF PDL AB

ALP 21d


PRFPPP

(e)


PRFPPP

100

80

60

40

20

0

Inte

grat

ed o

ptic

al d

ensit

y


lowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

DF PDL AB

Min 21d

(f)

PRF

PPP

OM

DMEM

(g)

PRF

PPP

OM

DMEM

(h)










6

5

4

3

2

1

0

Rela

tive e

xpre

ssio

n

DF PDL AB

RUNX2 7d

(a)

6

5

4

3

2

1

0

Relat

ive e

xpre

ssio

n

DF PDL AB

MGP 7d

(b)

6

5

4

3

2

1

0

Relat

ive e

xpre

ssio

n

DF PDL AB

RUNX2 14d



lowast

(c)

6

5

4

3

2

1

0

Relat

ive e

xpre

ssio

n

DF PDL AB

MGP 14d

lowastlowast

lowastlowastlowast

lowast

(d)

0

6

5

4

3

2

1

Relat

ive e

xpre

ssio

n

DF PDL AB

RUNX2 21d

lowastlowast

lowast


PRFPPP

(e)


PRFPPP

6

5

4

3

2

1

0

Relat

ive e

xpre

ssio

n

DF PDL AB

MGP 21d

(f)

62

37

PRF OM PPP DMEM

RUNX2

GAPDH

(g)

600

480

360

240

120

0

Inte

grat

ed d

ensit

y

PRF OM PPP DMEM

lowastlowastlowast

lowastlowastlowast

(h)



DF PDL AB



Fibrin coated

Untreated

(a)


Alizarin red S

DF PDL AB

Fibrin coated

Untreated

(b)


7d

PRF

200120583m

(a)

PRF

14d

Coll

(b)

PRF

14d

50120583m

lowast

(c)

14d

Coll

(d)



25 cm

(a)

PRF

(b)

(c) (d) (e)

(f) (g)

PRF

(h) (i)

(j) (k) (l)




4 Discussion











Acknowledgment


References













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


16

12

08

04

00

Opt

ical

den

sity

DF

1 2 3 4 5 6 7

Days of culture

PRFPPPDMEM

(a)

PRFPPPDMEM

12

08

04

00

Opt

ical

den

sity

PDL

1 2 3 4 5 6 7

Days of culture

(b)

12

08

04

00

Opt

ical

den

sity

AB

1 2 3 4 5 6 7

Days of culture

PRFPPPDMEM

(c)

25

20

15

10

5

0

Rel

num

ber o

f mig

rate

d ce

lls

DF PDL AB

PRFPPPDMEM



lowastlowast

(d)






30

25

20

15

10

5

0

Inte

grat

ed o

ptic

al d

ensit

y

DF PDL AB

lowast

lowast

ALP 7d

(a)

50

40

30

20

10

0

Inte

grat

ed o

ptic

al d

ensit

y

DF PDL AB

Min 7d

(b)

240

200

160

120

80

40

0

Inte

grat

ed o

ptic

al d

ensit

y

DF PDL AB

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

ALP 14d

(c)

75

60

45

30

15

0In

tegr

ated

opt

ical

den

sity

DF PDL AB

lowast lowast

lowastlowast


lowastlowastlowast


(d)

60

50

40

30

20

10

0

Inte

grat

ed o

ptic

al d

ensit

y


lowastlowastlowast

DF PDL AB

ALP 21d


PRFPPP

(e)


PRFPPP

100

80

60

40

20

0

Inte

grat

ed o

ptic

al d

ensit

y


lowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

DF PDL AB

Min 21d

(f)

PRF

PPP

OM

DMEM

(g)

PRF

PPP

OM

DMEM

(h)










6

5

4

3

2

1

0

Rela

tive e

xpre

ssio

n

DF PDL AB

RUNX2 7d

(a)

6

5

4

3

2

1

0

Relat

ive e

xpre

ssio

n

DF PDL AB

MGP 7d

(b)

6

5

4

3

2

1

0

Relat

ive e

xpre

ssio

n

DF PDL AB

RUNX2 14d



lowast

(c)

6

5

4

3

2

1

0

Relat

ive e

xpre

ssio

n

DF PDL AB

MGP 14d

lowastlowast

lowastlowastlowast

lowast

(d)

0

6

5

4

3

2

1

Relat

ive e

xpre

ssio

n

DF PDL AB

RUNX2 21d

lowastlowast

lowast


PRFPPP

(e)


PRFPPP

6

5

4

3

2

1

0

Relat

ive e

xpre

ssio

n

DF PDL AB

MGP 21d

(f)

62

37

PRF OM PPP DMEM

RUNX2

GAPDH

(g)

600

480

360

240

120

0

Inte

grat

ed d

ensit

y

PRF OM PPP DMEM

lowastlowastlowast

lowastlowastlowast

(h)



DF PDL AB



Fibrin coated

Untreated

(a)


Alizarin red S

DF PDL AB

Fibrin coated

Untreated

(b)


7d

PRF

200120583m

(a)

PRF

14d

Coll

(b)

PRF

14d

50120583m

lowast

(c)

14d

Coll

(d)



25 cm

(a)

PRF

(b)

(c) (d) (e)

(f) (g)

PRF

(h) (i)

(j) (k) (l)




4 Discussion











Acknowledgment


References













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


30

25

20

15

10

5

0

Inte

grat

ed o

ptic

al d

ensit

y

DF PDL AB

lowast

lowast

ALP 7d

(a)

50

40

30

20

10

0

Inte

grat

ed o

ptic

al d

ensit

y

DF PDL AB

Min 7d

(b)

240

200

160

120

80

40

0

Inte

grat

ed o

ptic

al d

ensit

y

DF PDL AB

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

ALP 14d

(c)

75

60

45

30

15

0In

tegr

ated

opt

ical

den

sity

DF PDL AB

lowast lowast

lowastlowast


lowastlowastlowast


(d)

60

50

40

30

20

10

0

Inte

grat

ed o

ptic

al d

ensit

y


lowastlowastlowast

DF PDL AB

ALP 21d


PRFPPP

(e)


PRFPPP

100

80

60

40

20

0

Inte

grat

ed o

ptic

al d

ensit

y


lowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

DF PDL AB

Min 21d

(f)

PRF

PPP

OM

DMEM

(g)

PRF

PPP

OM

DMEM

(h)










6

5

4

3

2

1

0

Rela

tive e

xpre

ssio

n

DF PDL AB

RUNX2 7d

(a)

6

5

4

3

2

1

0

Relat

ive e

xpre

ssio

n

DF PDL AB

MGP 7d

(b)

6

5

4

3

2

1

0

Relat

ive e

xpre

ssio

n

DF PDL AB

RUNX2 14d



lowast

(c)

6

5

4

3

2

1

0

Relat

ive e

xpre

ssio

n

DF PDL AB

MGP 14d

lowastlowast

lowastlowastlowast

lowast

(d)

0

6

5

4

3

2

1

Relat

ive e

xpre

ssio

n

DF PDL AB

RUNX2 21d

lowastlowast

lowast


PRFPPP

(e)


PRFPPP

6

5

4

3

2

1

0

Relat

ive e

xpre

ssio

n

DF PDL AB

MGP 21d

(f)

62

37

PRF OM PPP DMEM

RUNX2

GAPDH

(g)

600

480

360

240

120

0

Inte

grat

ed d

ensit

y

PRF OM PPP DMEM

lowastlowastlowast

lowastlowastlowast

(h)



DF PDL AB



Fibrin coated

Untreated

(a)


Alizarin red S

DF PDL AB

Fibrin coated

Untreated

(b)


7d

PRF

200120583m

(a)

PRF

14d

Coll

(b)

PRF

14d

50120583m

lowast

(c)

14d

Coll

(d)



25 cm

(a)

PRF

(b)

(c) (d) (e)

(f) (g)

PRF

(h) (i)

(j) (k) (l)




4 Discussion











Acknowledgment


References













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









6

5

4

3

2

1

0

Rela

tive e

xpre

ssio

n

DF PDL AB

RUNX2 7d

(a)

6

5

4

3

2

1

0

Relat

ive e

xpre

ssio

n

DF PDL AB

MGP 7d

(b)

6

5

4

3

2

1

0

Relat

ive e

xpre

ssio

n

DF PDL AB

RUNX2 14d



lowast

(c)

6

5

4

3

2

1

0

Relat

ive e

xpre

ssio

n

DF PDL AB

MGP 14d

lowastlowast

lowastlowastlowast

lowast

(d)

0

6

5

4

3

2

1

Relat

ive e

xpre

ssio

n

DF PDL AB

RUNX2 21d

lowastlowast

lowast


PRFPPP

(e)


PRFPPP

6

5

4

3

2

1

0

Relat

ive e

xpre

ssio

n

DF PDL AB

MGP 21d

(f)

62

37

PRF OM PPP DMEM

RUNX2

GAPDH

(g)

600

480

360

240

120

0

Inte

grat

ed d

ensit

y

PRF OM PPP DMEM

lowastlowastlowast

lowastlowastlowast

(h)



DF PDL AB



Fibrin coated

Untreated

(a)


Alizarin red S

DF PDL AB

Fibrin coated

Untreated

(b)


7d

PRF

200120583m

(a)

PRF

14d

Coll

(b)

PRF

14d

50120583m

lowast

(c)

14d

Coll

(d)



25 cm

(a)

PRF

(b)

(c) (d) (e)

(f) (g)

PRF

(h) (i)

(j) (k) (l)




4 Discussion











Acknowledgment


References













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


DF PDL AB



Fibrin coated

Untreated

(a)


Alizarin red S

DF PDL AB

Fibrin coated

Untreated

(b)


7d

PRF

200120583m

(a)

PRF

14d

Coll

(b)

PRF

14d

50120583m

lowast

(c)

14d

Coll

(d)



25 cm

(a)

PRF

(b)

(c) (d) (e)

(f) (g)

PRF

(h) (i)

(j) (k) (l)




4 Discussion











Acknowledgment


References













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


25 cm

(a)

PRF

(b)

(c) (d) (e)

(f) (g)

PRF

(h) (i)

(j) (k) (l)




4 Discussion











Acknowledgment


References













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



4 Discussion











Acknowledgment


References













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Acknowledgment


References













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article platelet-rich fibrin promotes...

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