Enzymatically induced mineralization of platelet-rich fibrin

Timothy E. L. Douglas,1,2 Volker Gassling,3 Heidi A. Declercq,4 Nicolai Purcz,3 Elzbieta Pamula,5

Havard J. Haugen,6 Safak Chasan,7 Eric L. W. de Mulder,8 John A. Jansen,1

Sander C. G. Leeuwenburgh1

1Department of Biomaterials, Radboud University Medical Center Nijmegen, P.O. Box 9101, 6500 HB Nijmegen,

The Netherlands2Polymer Chemistry and Biomaterials (PBM) Group, Department of Organic Chemistry, University of Ghent, Krijgslaan 281

S4, B-9000 Ghent, Belgium3Clinic of Oral and Maxillofacial Surgery, University Hospital Schleswig-Holstein, 24105 Kiel, Germany4Department of Basic Medical Science–Histology Group, Ghent University, De Pintelaan 185 (6B3), B-9000 Ghent, Belgium5Department of Biomaterials, Faculty of Materials Science and Ceramics, AGH University of Science and Technology, Al.

Mickiewicza 30, 30-059 Krakow, Poland6Department of Biomaterials, Institute for Clinical Dentistry, University of Oslo, PO Box 1109 Blindern, NO-0317 Oslo, Norway7Department of Molecular Biology and Genetics, Middle East Technical University, 06531 Ankara, Turkey8Department of Orthopedics, Radboud University Medical Center Nijmegen, The Netherlands

Received 18 December 2011; accepted 5 January 2012

Published online in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.34073

Abstract: Membranes of the autologous blood-derived bio-

material platelet-rich fibrin (PRF) were functionalized by

incorporation of alkaline phosphatase (ALP), an enzyme

involved in mineralization of bone, and subsequently incu-

bated in calcium glycerophosphate (CaGP) solution to induce

PRFs mineralization with calcium phosphate (CaP) to improve

PRFs suitability as a material for bone replacement. Incorpo-

rated ALP retained its bioactivity and induced formation of

CaP material within PRF membranes, as confirmed by SEM,

EDS, FTIR, and von Kossa staining. The mass percentage at-

tributable to CaP was quantified by lyophilization and mea-

surement of the remaining mass fraction as well as by TGA.

Cytocompatibility tests (LDH, MTT, and WST) with SAOS-2

cells showed that mineralized PRF did not release substances

detrimental to cell vitality. Live/dead staining and SEM

showed that mineralized PRF was colonized by cells. The

results show that hydrogel biomaterials such as PRF can be

mineralized through functionalization with ALP. VC 2012 Wiley

Periodicals, Inc. J Biomed Mater Res Part A: 00A:000–000, 2012.

Key Words: alkaline phosphatase, mineralization, hydrogel,

bone tissue engineering, platelet-rich fibrin

How to cite this article: Douglas TEL, Gassling V, Declercq HA, Purcz N, Pamula E, Haugen HJ, Chasan S, de Mulder ELW, JansenJA, Leeuwenburgh SCG. 2012. Enzymatically induced mineralization of platelet-rich fibrin. J Biomed Mater Res Part A2012:00A:000–000.

INTRODUCTION

Discovered by Choukroun in 2000, platelet-rich fibrin(PRF) is a blood-derived fibrin-based material originallydesigned for and applied in oral and maxillofacial surgery,particularly sinus-lifts,1–5 though it has also been used formiddle ear surgery6 and peri-implant defect filling.7 PRF isobtained by centrifugation of patient’s blood without anti-coagulant, which results in a fibrin clot in the middle ofthe centrifugation tube, in which platelets are trapped.8

Several of these clots can be compressed to form a mem-brane from which growth factors are released slowly overthe course of several days.9,10 Cell biological characteriza-tion of PRF has demonstrated the biocompatibility of PRF

and its ability to support the proliferation of a range ofcell types.10–13

Mineralization of PRF is anticipated to lead to a numberof advantages from both clinical and basic science points ofview. First, the mechanical reinforcement of the gel as aresult of mineralization could improve ease of handling forclinicians and the stability of PRF as a barrier membrane inguided bone regeneration, and also potentially, in the future,enable new applications for PRF as a scaffold material whichrequire higher mechanical strength. Second, as stiffer14,15 androugher16 surfaces are known to promote differentiation ofcells toward the osteoblastic phenotype, mineralization isexpected to make PRF more bone-friendly. Furthermore, the

Correspondence to: S. C. G. Leeuwenburgh; e-mail: s.leeuwenburgh@dent.umcn.nl

Contract grant sponsor: Dutch organization Agentschap NL in the framework of the IOP program ‘‘Self Healing Materials’’; contract grant

number: SHM08717 (Self-healing composites for bone substitution)

Contract grant sponsors: T.E.L. Douglas - Postdoctoral Fellowship of Research Foundation Flanders (FWO): H.A. Declercq - Postdoctoral BOF-

Mandate (Ghent University).

VC 2012 WILEY PERIODICALS, INC. 1

presence of mineral in fibrin may increase resistance to deg-radation, aid protein adsorption and cell attachment, andhelp to induce bone formation, as reported for intramuscularimplantation of fibrin-CaP composites.17

The enzyme alkaline phosphatase (ALP) supports miner-alization of bone in vivo by cleavage of phosphate fromorganic phosphate. ALP has been successfully incorporatedinto polymeric hydrogels such as polyHEMA,18–20 colla-gen,21,22 and artificial self-assembling peptide amphiphilegels23 to cause mineralization. However, there are no publi-cations dealing with the application of such mineralizedhydrogels (such as enzymatically mineralized fibrin-basedmaterials) as bone tissue engineering scaffolds.

The aim of this study was the mineralization of PRF mem-branes by incorporation of ALP, which was added to blooddirectly before centrifugation on the assumption that it would,to some extent, be entrapped in the fibrin clot during centrifu-gation and remain in the PRF membrane after pressing outfluid (Fig. 1). This assumption was based on the fact thatsmall growth factors are released slowly from PRF, suggestingthat the PRF matrix impedes their diffusion. As ALPs molecu-lar weight has been reported to be 185 kDa24 and thus it is alarger molecule than growth factors, it was predicted that ALPdiffusion would be similarly hindered. For comparison, growthfactors present in PRF, such as platelet-derived growth factor(PDGF), vascular endothelial growth factor (VEGF), and trans-forming growth factor beta 1 (TGF-b1),25 have molecularweights of 32 kDa,26 46kDa,27 and 25 kDa,28 respectively.

This study is aimed to determine (a) if ALP can beincorporated into PRF membranes, retain its bioactivity,

and induce mineralization; (b) the nature, amount, and dis-tribution of the mineral thus formed; (c) the effect of min-eralization on PRF morphology, and (d) if enzymaticallymineralized PRF is cytocompatible. To this end, ALP-con-taining PRF membranes were incubated in a solution con-taining calcium glycerophosphate (CaGP), which served as asource of organic phosphate and calcium ions. CaGP dif-fused into the membrane, where under the action of ALP,phosphate was released which reacted with calcium ions toform calcium phosphate (CaP). Subsequently, the mineral-ized PRF membranes were analyzed physicochemically[using Fourier-Transfer Infrared (FTIR) spectroscopy andFTIR microscopy, scanning electron microscopy (SEM) andenergy dispersive spectroscopy (EDS), and by quantificationof the remaining mass percentage postlyophilization as wellas thermogravitational analysis (TGA)], histologically (usingvon Kossa staining), and biologically (by culturing SAOS-2cells in vitro and applying standard cytocompatibility tests(LDH, MTT, and WST) and imaging by live/dead stainingand SEM).

MATERIALS AND METHODS

Incorporation of ALP into PRF and subsequentmineralizationPRF membranes were produced as described previously.29,30

Briefly, patients donated 40 mL of their whole blood in10 mL tubes without anticoagulant (Vacuette 455092, GreinerBio-One GmbH, Frickenhausen, Germany). Ethical approval(A 118/07) was issued by the Ethics Commission, Christian-Albrechts-University of Kiel, Germany. Blood samples were

FIGURE 1. Schematic description of incorporation of alkaline phosphatase (ALP) into PRF membranes. [Color figure can be viewed in the online

issue, which is available at wileyonlinelibrary.com.]

2 DOUGLAS ET AL. ENZYMATICALLY INDUCED MINERALIZATION OF PRF

immediately centrifuged for 12 min at 400�g. After activa-tion of the coagulation cascade by contact of blood plateletswith the tube walls, a fibrin clot was obtained in the middlebetween the acellular plasma at the top and red blood cellsat the bottom of the receptacle. The PRF clot was separatedfrom the red blood cells using a sterile syringe and scissorsand then transferred onto a sterile compress. A fibrin mem-brane of thickness under 0.5 mm was obtained by squeez-ing serum out of the PRF clot manually. ALP was incorpo-rated into PRF clots by addition of 1 mL of 20 mg/mL ALP(Sigma, P7640, from bovine intestinal mucosa) in phosphatebuffered saline (PBS) to 10 mL of blood before centrifuga-tion (Fig. 1). Discs of diameter 5 mm were cut out of PRFmembranes.

Release of ALP from PRFPRF samples with or without added ALP were weighed andincubated in 1.5 mL of ultrapure water (Milli-Q). ALP activ-ity in the Milli-Q release medium was determined 30, 60,90, 150, 210, and 270 min after the start of incubationusing a standard ALP activity assay31 with modifications. Inbrief, 500 lL of incubation medium (i.e., water) containingreleased ALP was transferred to a cuvette. A total of 250 lLof a substrate solution consisting of 5 mM p-nitrophenyl-phosphate disodium salt (Sigma, P5994) in 0.5M alkalinebuffer was added. This buffer was prepared by diluting1.5M alkaline buffer (Sigma, A9226) with Milli-Q in the vol-umetric ratio 1:2. After �3 min incubation, the reaction wasstopped by addition of 250 lL 0.3M NaOH (aq). Absorbancewas measured at 405 nm using an Uvikon UV-Vis spectro-photometer (Bio-Tek Instruments). A calibration curve ofALP in water with concentrations ranging from 0 to0.03 mg/mL served as a reference to relate activity toenzyme concentration. This allowed calculation of the massof ALP released, which was then divided by PRF samplemass to obtain mass of ALP released per unit mass PRF,expressed in lg ALP/mg PRF.

Mineralization and characterization of membranesALP-containing PRF and ALP-free PRF were incubated for 3days in 0.1M Ca-GP (Spectrum, CA, USA) in Milli-Q. After-wards, samples were rinsed three times with Milli-Q andincubated for 24 h in Milli-Q before further experiments.

Physicochemical characterization of PRF membranesThe mass percentage postlyophilization was calculated as(n ¼ 3): (weight after lyophilization/weight before lyophili-zation)*100. Lyophilization was performed at �50�C for48 h to maximize the drying efficiency. Lyophilization is astandard method to dry hydrogel samples.32,33

Bearing in mind that lyophilization might not lead to acompletely dry product, and that mineral formation mighthinder lyophilization, TGA was performed using a Hi-ResTGA 2950 Thermogravimetric Analyzer (TA Instruments).Samples (n ¼ 3) were heated from 30 to 800�C at a rate of10�C/min under constant monitoring of remaining weightpercentage.

Elemental composition of the samples was studied byEnergy Dispersive X-ray Spectroscopy (EDS, EDAX, USA)connected to a scanning electron microscope (SEM) (NovaNanoSEM 200, FEI, USA; accelerating voltage 18 kV) at amagnification of 6000�. Before the analysis, samples werelyophilized for 48 h before examination and sputter coatedwith a thin carbon layer to make them conductive.

FTIR and FTIR microscopic imagingThe molecular structure of the membranes was examinedafter mineralization experiments using Attenuated TotalReflectance Fourier-Transform Infrared Spectroscopy (ATR-FTIR, Spectrum One). Samples were lyophilized for 48 hbefore examination.

The FTIR microscope (Spotlight 400, Perkin-Elmer, Oslo,Norway) images of PRF membranes with and without addedALP in the wet, unlyophilized state were recorded in trans-mission mode with pixel size of 6.2 lm at a resolution of8 cm�1, with 25 scans per pixel, and an interferometerspeed of 1.0 cm/s. FTIR images (493 � 375 lm2) consistingof 4880 single spectra displayed the average absorbance in-tensity of all wavenumbers ranging from 1500 to 650 cm�1

or the intensity at a single wavenumber of 1030 cm�1. Sam-ples were chopped into fine pieces and pressed gentlybetween glass slides to a thickness of 10 lm to allow trans-mission images to be obtained.

Histological preparation and stainingMineral distribution within PRF membranes was visualizedby histological sectioning followed by von Kossa staining forphosphate deposits according to standard protocols. Briefly,samples were fixed in 4% formaldehyde overnight. Subse-quently, samples were incubated in 70% ethanol for 5 hand then for 1 h in each of the following solutions: 80, 90,and 96% ethanol, pure methyl benzolate, 1% celloidine inmethyl benzolate, 2% celloidine in methyl benzolate, puremethyl benzolate, chloroform, and histoclearTM. Thereafter,samples were incubated in paraffine for 2 h and in new par-affine for 4 h.

Samples were then embedded in parrafine and sliced. Atotal of 5-lm sections were made and immobilized on glass

FIGURE 2. Release profiles of ALP from PRF membranes with added

ALP (top, diamonds) and without added ALP (bottom, triangles).

[Color figure can be viewed in the online issue, which is available at

wileyonlinelibrary.com.]

ORIGINAL ARTICLE

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2012 VOL 00A, ISSUE 00 3

slides. After deparaffination, samples were subjected tovon Kossa staining. Briefly, slices were placed in demine-ralized water and exposed to 5% silver nitrate solution for

30 min under UV irradiation. After rinsing in demineral-ized water, slices were exposed to 2% sodium thiosulfatefor 5 min and then rinsed with running water for 10 min.

FIGURE 3. SEM Images of PRF membranes with (a,c) and without (b,d) added ALP. Scale bar 1 lm (a,b) or 100 nm (c,d).

FIGURE 4. EDS analysis of PRF membranes with (left) and without added ALP (right). [Color figure can be viewed in the online issue, which is

available at wileyonlinelibrary.com.]

4 DOUGLAS ET AL. ENZYMATICALLY INDUCED MINERALIZATION OF PRF

After rinsing in demineralized water, slices were exposedto a 0.5% Nuclear Fast Red in 5% aluminium sulfate solu-tion for 10 min. After rinsing in demineralized water, sliceswere dehydrated and covered with glass coverslips formicroscopic analysis.

Cell biological characterizationCell biological characterization was performed using the sar-coma osteogenic SAOS-2 cell line. Cytocompatibility wasassessed using standard cell vitality assays [lactate dehydro-genase (LDH), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT), and water soluble tetrazolium salts(WST)] as described in detail in previous publications of theauthors.29,34–37 Briefly, circular PRF membrane samples ofdiameter 5 mm containing ALP and samples without ALPwere incubated in 0.1M CaGP for 3 days and then washedtwice in PBS. Subsequently, all samples (in duplicate) wereincubated for 10 min in 1 mL serum-free cell culturemedium (DMEM GlutaMax High Glucose). No serum wasadded as it contains LDH. This serum-free cell culturemedium in which incubation took place, hereafter referredto as eluate, was removed and retained for LDH and MTTtests. The incubation was then repeated in fresh serum-freecell culture medium but for 1 h after which the eluate wasremoved and retained, fresh serum-free cell culture mediumadded, incubation performed for 24 h, and the eluateremoved and retained. Membranes were then seeded with10,000 cells for the WST test and incubated in cell culturemedium (DMEM GlutaMax High Glucose containing 10%fetal bovine serum, 0.1 mM sodium pyruvate, and 1% Peni-cillin-Streptomycin) for 7 days with medium change every2 days after which the WST test was performed.

FIGURE 5. (a) FTIR spectra of lyophilized PRF with added ALP (top,

black) and PRF without added ALP (bottom, blue) after incubation in

mineralization solution for 3 days. A band at �1030 cm�1 (*) and a

double band at �600 and 560–570 cm�1 (#) were observed for PRF

with ALP. [Color figure can be viewed in the online issue, which is

available at wileyonlinelibrary.com.]

FIGURE 6. FTIR microscopy images of PRF membranes. A: Average absorbance intensity from area containing two large PRF membrane pieces

with added ALP (top left, center). B: Absorbance intensity at wave number 1030 cm�1 from same image area as A. C: Average absorbance inten-

sity of area containing two large PRF membrane pieces without added ALP (top left, center) with similar shapes to those shown in (A). D: Ab-

sorbance intensity at wave number 1030 cm�1 from same image area as (C). [Color figure can be viewed in the online issue, which is available

at wileyonlinelibrary.com.]

ORIGINAL ARTICLE

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2012 VOL 00A, ISSUE 00 5

To obtain SEM images of cells on PRF, cells were seededand cultivated in the same fashion as for the WST test, butfor 5 days. Cells on PRF were washed with PBS then fixedby dilution with 2.5% glutaraldehyde and 2.5% paraformal-dehyde in 0.1M cacodylate buffer (four changes over 5 min),followed by incubation in undiluted fixative for 1 h at roomtemperature. After washing 3 � 1 min in ice-cold 0.1M cac-odylate buffer, cells on PRF were subsequently postfixed in

2% OsO4 in 0.1M cacodylate buffer for 30 min at room tem-perature in a fume hood. After dehydration by incubation in50%, 75%, 85%, and 2 � 95% ethanol solutions for 30 minper solution, critical point drying was performed using aBAL-TEC CPD 030 Critical Point Dryer. This was followed bygold sputtering using a JEOL JFC-1200 Fine Coater beforeSEM using a JEOL JSM-5600LV microscope.

For the LDH and MTT test, 5000 cells were seeded intowells of 96-well cell culture plates (Nunc, Denmark). Eluateretained after 10 min, 1 h, and 24 h incubation was addedto cells: 150 lL for the LDH test and 100 lL for the MTTtest. In the LDH test, cells incubated in 1% Triton X-100and cells incubated in serum-free cell culture mediumserved as positive and negative controls, respectively. In theMTT test, cells incubated in serum-free cell culture mediumserved as high and low controls.

Live/dead staining (calcein AM/propidium iodide) wasperformed to evaluate cell viability. After rinsing the cell-seeded membranes with PBS, the supernatant was replacedby 1 mL PBS supplemented with 2 lL (1 mg/mL) calceinAM (Anaspec, USA), and 2 lL (1 mg/mL) propidium iodide(Sigma). Cultures were incubated for 10 min at room tem-perature, washed twice with PBS, and evaluated by fluores-cence microscopy (Olympus inverted Research SystemMicroscope, CellM software). Evaluations were performed 1and 5 days postseeding.

RESULTS

Release of ALP from PRFThe release profiles of ALP from PRF are shown in Figure 2.Considerably, more ALP is released from samples to whichALP is added (1.48 6 0.14 lg/mg PRF after 6 h in compari-son to 0.09 6 0.13 lg/mg PRF for samples without addedALP). As regards release kinetics, a burst release wasobserved for PRF with added ALP, where �78% of all ALPreleased is liberated after 30 min and �91% after 60 min,followed by a gradual decrease in release rate until a pla-teau is reached after 300 min. In the case of PRF withoutALP, all ALP released was liberated within 30 min.

Physicochemical analysesSEM images of PRF membranes with and without ALP areshown in Figure 3. Samples containing ALP [Fig. 3(a,c)]showed a markedly different morphology. At a magnification

FIGURE 7. Von Kossa staining of PRF membranes. Mineral is stained

brown. Top and middle: with added ALP; bottom: without added ALP.

Scale bar: 200 lm. [Color figure can be viewed in the online issue,

which is available at wileyonlinelibrary.com.]

FIGURE 8. Mass percentage of PRF with and without added ALP

remaining after lyophilization. [Color figure can be viewed in the

online issue, which is available at wileyonlinelibrary.com.]

6 DOUGLAS ET AL. ENZYMATICALLY INDUCED MINERALIZATION OF PRF

of 50,000�, homogenously distributed mineral-like depositsof length 100–200 nm were visible in the sample with ALP[Fig. 3(c)], which were absent in the sample without ALP[Fig. 3(d)].

EDS spectra of PRF membranes with and without addedALP are shown in Figure 4. Samples containing ALP dis-played peaks for calcium and phosphorous, which wereabsent in samples without ALP.

FTIR spectra of PRF membranes with and without ALPincubated for 3 days in 0.1M CaGP are shown in Figure 5.PRF containing ALP displayed a band at �1030 cm�1 and adouble band at �600 and 570–60 cm�1, showing the pres-ence of CaP.38 These bands were absent in the spectrum ofALP-free PRF. In the spectrum of PRF with ALP, bands verysimilar in intensity to those in the the spectrum of PRFwithout ALP were observed in the region 1800–1200 cm�1,suggesting that mineral was not the dominant phase in themineralized PRF.

FTIR microscopy images of pieces of PRF membraneswith and without added ALP are shown in Figure 6. Com-parison of average absorbance intensity of all wave numbersranging from 1500 to 650 cm�1 of PRF pieces with addedALP [Fig. 6(a)] as well as the absorbance intensity at1030 cm�1 [Fig. 6(b)] revealed that CaP was distributed

homogeneously in the large pieces, which were the moststrongly mineralized regions. In the vicinity of the largepieces, the presence of many much smaller PRF pieces canbe observed, which are formed during sample preparation,i.e., cutting the mineralized PRF membrane. The fact thatthese are more prominent in the 1030 cm�1 image suggeststhat they predominantly consist of CaP. Images of averageabsorbance intensity [Fig. 6(c)] and absorbance intensity at1030 cm�1 [Fig. 6(d)] of PRF pieces without added ALPrevealed the absence of CaP, apart from in two isolatedregions. The regions of higher intensity in Figure 6(c) maythus be ascribed to fibrin.

Von Kossa staining of mineral depositsLight microscopy images of disc-shaped PRF membraneswith and without added ALP incubated for 3 days in 0.1MCaGP (aq) and subjected to von Kossa staining are shownin Figure 7. The membrane without ALP was devoid ofbrown staining characteristic for phosphate deposits.Membranes containing ALP stained positive for phos-phate. Staining of cross-sections perpendicular to circularsurface of discs revealed mineral formation throughoutthe membranes.

FIGURE 9. Thermogravitational analysis (TGA). Curves showing decrease in weight percentage as a function of temperature for PRF with (a)

and without (b) added ALP. (c) Weight percentage remaining after final temperature of 800�C is reached. [Color figure can be viewed in the

online issue, which is available at wileyonlinelibrary.com.]

ORIGINAL ARTICLE

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2012 VOL 00A, ISSUE 00 7

Quantification of mass percentage after lyophilizationand TGAThe mass percentages after lyophilization of PRF mem-branes with and without added ALP are shown in Figure 8.The values were 15.3% 6 0.6% with and 12.5% 6 1.0%without added ALP. A student’s t-test revealed the differen-ces to be statistically significant (p ¼ 0.04, <0.05).

Representative TGA curves of PRF with and withoutadded PRF are shown in Figure 9(a) and (b), respectively.Samples without added ALP were completely destroyed byheating to 800�C, whereas samples with added ALP retaineda certain mass fraction, which was 20.7% 6 8.6% for min-eralized PRF and 0.5% 6 2.7% for nonmineralized PRF [Fig.9(c)]. Heating in the range up to 200�C caused weight per-centage decreases, which were 7.6% 6 1.2% for PRF withadded ALP and 8.3% 6 1.1% for PRF without added ALP.At 150�C and 200�C, the values were 9.1% 6 1.5% and8.3% 6 1.1% and 9.7% 6 1.6%, and 10.2% 6 0.8%,respectively. The differences between the two groups werenot significant in the range 30–200�C (student’s t-test).

Cell biological characterization: cytocompatibility testsResults of the LDH, MTT, and WST tests are shown in Figure10(a), (b), and (c), respectively. The LDH test, which is ameasure of cell lysis, revealed that PRF with or withoutadded ALP displayed a similar absence of cytotoxicity to thenegative control. The MTT test, a measure of mitochondrialactivity, showed that cells cultured in eluate from PRF withor without added ALP showed similar mitochondrial activityto controls. WST test results revealed considerably lowerproliferation on PRF with added ALP than on PRF withoutadded ALP and tissue culture polystyrene.

Cell biological characterization: live/deadstaining and SEM imagingLive/dead fluorescence microscopy images of SAOS-2 cellson PRF membranes with and without added ALP are shownin Figure 11. Viable cells, stained green, were visible on bothmembranes after 1 day and 5 days. As a general observation,greater cell numbers were seen on PRF without added ALP.This difference was particularly evident after 1 day [Fig. 11(a,b)].Some dead cells were seen on all samples.

SEM images of SAOS-2 cells on PRF with and withoutadded ALP are shown in Figure 12. As a general observa-tion, more cells were present on PRF without added ALP,which formed a confluent layer [Fig. 12(b)], whereas onPRF with added ALP, single cells were seen which displayeda well-spread morphology [Fig. 12(a)] and numerous exten-sions [Fig. 12(a,c,e)]. Higher magnification images of cellsand substrates reveal mineral on PRF with added ALP inthe form of nodules fixed to the surface and clusters on topof the surface [Fig. 12(c,e)]. These features were absent onPRF without added ALP [Fig. 12(d,f)].

DISCUSSION

As stated in the introduction, this study aimed to determine(a) if ALP can be incorporated into PRF membranes, retainits bioactivity, and induce mineralization; (b) the nature,amount, and distribution of the mineral thus formed; (c) theeffect of mineralization on PRF morphology, and (d) if enzy-matically mineralized PRF are cytocompatible.

ALP is known to be involved in bone formation and min-eralization processes.39 As a biomaterial component, ALPhas been applied as a coating for metallic implants for bonecontact31,40,41 and covalently linked to fibrin scaffolds toincrease mineral deposition in vitro and bone formationin vivo in a mouse calvarial defect.42

As mentioned in the introduction, mineralization ofhydrogel materials has been achieved by incorporation ofALP followed by soaking in a solution containing calciumions and glycerophosphate as a substrate for ALP, whichcleaves off phosphate which is then free to react with cal-cium to form insoluble CaP within the gel.

ALP-induced mineralization was demonstrated by theformation of mineral-like deposits as revealed by SEM (Fig.3), the presence of Ca and P peaks in EDS spectra (Fig. 4),bands characteristic for CaP in FTIR spectra (Fig. 5), andpositive von Kossa staining (Fig. 7), exclusively in mem-branes with added ALP.

FIGURE 10. Biocompatibility experiments performed using the cell

line SAOS-2. PRF membranes with and without added ALP incubated

for 3 days in mineralization solution. (a) Cytotoxicity of eluate from

PRF membranes collected after 10 min, 1 h, and 24 h to SAOS-2 cells

after 1 day of culture as measured by LDH test. (b) Mitochondrial ac-

tivity of SAOS-2 cells cultured in eluate from PRF membranes col-

lected after 10 min, 1 h, and 24 h as measured by MTT test. (c)

Proliferation of SAOS-2 cells cultured on PRF membranes with and

without added ALP as measured by WST test. Tissue culture polysty-

rene served as a control. [Color figure can be viewed in the online

issue, which is available at wileyonlinelibrary.com.]

8 DOUGLAS ET AL. ENZYMATICALLY INDUCED MINERALIZATION OF PRF

ALP-induced mineral-like deposits observed by SEMwere homogenously distributed over the PRF surface andwere of approximate diameter 100–200 nm [Fig. 3(a,c)],which is similar to the ALP-induced deposits seen on colla-gen22 and peptide amphiphile gels,23 whereas 1–4 lmdeposits were seen on polyHEMA.18,19 The observed differ-ence in morphology is the result of the ALP-induced mineralformation. Soluble CaGP in the form of calcium ions andglycerophosphate diffuses into the PRF membrane, whereentrapped ALP cleaves phosphate from glycerophosphate.These then react to form insoluble CaP, which is depositedinside the polymer network within the membrane. Depositson the surface are detected by SEM. ALP-free PRF displayeda smooth morphology [Fig. 3(b,d)] similar to that observedin our previous studies.29 The homogeneous distribution ofthe deposits [Fig. 3(a,c)] suggests homogeneous distributionof the ALP on the PRF surface.

With the aim of increasing the mineral content of fibrin,another approach is the incorporation of preformed CaPparticles. In publications of the group of Daculsi, fibrin gluehas been mixed with CaP granules of diameter 1–2 mmbefore implantation,43–46 which led to improved handling,spontaneously induced bone formation after implantation insheep muscle,17 and successful application for sinus lifts ina sheep model.47 Enzymatic mineralization offers an alterna-tive to addition of CaP particles and enables a more homo-geneous mineralization. The mineral distribution on thesurface of ALP-enriched PRF in this study appears morehomogeneous than that of CaP particles mixed into fibrin

glue.48 In addition, the mineral formed by ALP inside PRFby interaction with its chemical environment was shown tobe in the nanorange [see Fig. 3(a,c)], and thus smaller thanthe 1–2mm particles added by Dalcusi and coworkers tofibrin glue.17,43–45,48

FTIR analysis showed that the mineral formed displayedbands at �1030 cm�1 and a double band at 600 and 560–570 cm�1 (Fig. 5). These bands suggest the presence ofapatite38; however, the presence of other CaP types is notexcluded, particularly as the type of CaP formed in hydrogelscontaining ALP in literature differs. Formation of hydroxyapa-tite was reported on polyHEMA gels18,19 and artificial peptideamphiphile gels.23 Banks et al. and Doi et al. found that apatiteformed on collagen sheets soaked in ALP,49,50 whereas Yamau-chi et al. reported that ALP in collagen sheets formed a mix-ture of hydroxyapatite and amorphous CaP.22

The FTIR microscopy images show that CaP was distrib-uted homogeneously in PRF membrane pieces with addedALP [Fig. 6(a,b)]. CaP was absent in PRF without added ALP[Fig. 6(c,d)] except for two isolated regions, where it can bespeculated that the local concentration of ALP (from bloodserum) is higher. Homogeneous distribution of CaP in PRFwith added ALP is in agreement with the results of vonKossa staining (Fig. 7), which was negative for PRF withoutadded ALP but showed distribution of CaP throughout thethickness of the PRF membrane with added ALP.

TGA showed that the mass percentage remaining at800�C was 20.7% 6 8.6% for PRF with added ALP and0.5% 6 2.7% for PRF without ALP [Fig. 9(c)]. In view of

FIGURE 11. Live/dead fluorescence microscopy images of SAOS-2 cells on PRF membranes with (a,c) and without added ALP (b,d) after 1 day

(a,b) and 5 days (c,d). Scale bar: 200 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

ORIGINAL ARTICLE

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2012 VOL 00A, ISSUE 00 9

the aformentioned results demonstrating ALP-mediated min-eralization of PRF, the whole of the mass percentage remain-ing at 800�C in mineral samples, �20%, can be ascribed toCaP mineral, which can withstand thermal testing at 800�C.51

This is in agreement with the difference in mass percentagepostlyophilization between mineralized (15.3% 6 0.6%) andunmineralized PRF (12.5% 6 1.0%) in Figure 8, suggesting amineral mass percentage of �18% in mineralized PRF. TGAalso revealed mass losses in the region 30–200�C, which maybe ascribed to water and show that lyophilization does notyield a perfectly dry product. The difference between thegroups in this region was not significant, meaning that thepresence of mineral does not significantly hinder mineraliza-tion and that comparison of mass remaining postlyophiliza-tion was a reliable measure of mineral formed in this study.

The suitability of PRF with and without added PRF ascell carriers was demonstrated by cytocompatibility assays(Fig. 10), Live/dead staining and fluorescence microscopy

(Fig. 11), and SEM imaging (Fig. 12), which proved the pres-ence of viable SAOS-2 cells on both mineralized and unmin-eralized PRF.

Cytocompatibility investigations with SAOS-2 cellsshowed that mineralized PRF did not release substanceswhich hindered cell growth [Fig. 10(a,b)]. WST test resultsshowed that proliferation was inferior on PRF with addedALP [Fig. 10(c)], which is in agreement with the results of theSEM investigations, where more cells were present on PRFwithout ALP in confluent layers [Fig. 12(b)], whereas fewercells were observed on PRF with added ALP [Fig. 12(a)].SEM also revealed mineral deposits on PRF containing ALP[Fig. 12(a,c,e)], which were absent on PRF without ALP[Fig. 12(d,f)]. This is in agreement with the results of theexperiments carried out to evaluate mineralization in theabsence of cells.

There are several possible explanations for the lowerproliferation on PRF with added ALP. As mentioned earlier,

FIGURE 12. SEM images of SAOS-2 cells after 5 days cultivation on PRF membranes with (a,c,e) and without added ALP (b,d,f). Magnifications:

a,b: 1000�; c,d: 5000�; e,f: 10,000�. Scale bars: a,b: 10 lm; c,d: 5 lm; e,f: 1 lm.

10 DOUGLAS ET AL. ENZYMATICALLY INDUCED MINERALIZATION OF PRF

the CaP formed by ALP may release Ca2þ ions, resulting inconcentrations toxic to cells, particularly if soluble, noncrys-talline CaP phases are present. Second, CaP may be poorerat promoting adhesion and cell proliferation than fibrinbecause of the abundant presence of RGD peptide sequen-ces in fibrin that modulate cell adhesion. Moreover, poorerosteoblastic cell growth on amorphous CaP has beenreported.52 Finally, it is known for many cell types that pro-liferation declines when differentiation begins.16,53 The addi-tion of ALP and subsequent deposition of CaP onto thesurfaces of the PRF membrane may stimulate differentiationand decrease proliferation. In that respect, the observedincrease in surface roughness due to mineralization of thePRF might also have contributed to reduced proliferation.16

As expected, more ALP was released from PRF sampleswith added ALP (Fig. 2). The relatively small signal fromPRF samples without added ALP is probably due to thepresence of various isoenzymes of ALP in blood serum54,55

which may become entrapped during formation of the PRFmembrane. It should be stressed that the activity of bloodserum ALP isoenzymes is unknown and may differ fromthat of the added ALP. Thus, the mass of ALP calculated forthe PRF samples without added ALP actually represents anequivalent value of added ALP. Release from samples with-out added ALP did not increase appreciably after 30 min,whereas a burst release was observed for PRF with addedALP, followed by a gradual decrease in release rate until aplateau is reached after 300 min. This suggests that all ALPreleased is liberated within 6 h.

In contrast to the release profiles of ALP in this study,Dohan Ehrenfest et al.9 and He et al.10 reported slowrelease of growth factors PDGF, VEGF, and TGF-b1 over 168h and 28 days, respectively, whereas Su et al.56 investigatedrelease of growth factors and found that no plateau valueswere reached after 300 min. First of all, it should be empha-sized that the amounts of growth factors released in thesestudies are an order of magnitude lower than the ALPamounts released in this study. In view of the high molecu-lar weight (185 kDa) and large size of ALP24 relative to thelow molecular weight (typically 15–30 kDa) and size ofgrowth factors, the fast release of ALP as observed in thisstudy relative to the slow release of several growth factorsobserved previously suggests these larger ALP moleculesare not bound more strongly to fibrin by electrostatic inter-actions or more strongly physically entrapped within thefibrin network.

CONCLUSIONS

This work showed the feasibility of inducing mineral forma-tion in PRF by incorporation of ALP during hydrogel forma-tion and subsequent incubation in CaGP solution, resultingin cytocompatible membranes with nanoparticles of CaPhomogeneously distributed on the PRF surface which consti-tuted �20% of the mass after lyophilization.

ACKNOWLEDGMENTS

The authors thank Renaat Dasseville, Gabi Nessenius, and G.Otto for technical assistance.

REFERENCES1. Choukroun J, Diss A, Simonpieri A, Girard MO, Schoeffler C,

Dohan SL, Dohan AJ, Mouhyi J, Dohan DM. Platelet-rich fibrin

(PRF): A second-generation platelet concentrate. Part V: Histologic

evaluations of PRF effects on bone allograft maturation in sinus

lift. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;101:

299–303.

2. Charrier JB, Monteil JP, Albert S, Collon S, Bobin S, Dohan

Ehrenfest DM. Relevance of Choukroun’s platelet-rich fibrin (PRF)

and SMAS flap in primary reconstruction after superficial or sub-

total parotidectomy in patients with focal pleiomorphic adenoma:

A new technique. Rev Laryngol Otol Rhinol 2008;129:313–318.

3. Mazor Z, Horowitz RA, Del Corso M, Prasad HS, Rohrer MD,

Dohan Ehrenfest DM. Sinus floor augmentation with simultane-

ous implant placement using Choukroun’s platelet-rich fibrin as

the sole grafting material: A radiologic and histologic study at 6

months. J Periodontol 2009;80:2056–2064.

4. Simonpieri A, Del Corso M, Sammartino G, Dohan Ehrenfest DM.

The relevance of Choukroun’s platelet-rich fibrin and metronida-

zole during complex maxillary rehabilitations using bone allo-

graft. Part II: Implant surgery, prosthodontics, and survival.

Implant Dent 2009;18:220–229.

5. Simonpieri A, Del Corso M, Sammartino G, Dohan Ehrenfest DM.

The relevance of Choukroun’s platelet-rich fibrin and metronidazole

during complex maxillary rehabilitations using bone allograft. Part

I: A new grafting protocol. Implant Dent 2009;18:102–111.

6. Braccini F, Tardivet L, Dohan Ehrenfest DM. [The relevance of

Choukroun’s Platelet-Rich Fibrin (PRF) during middle ear surgery:

Preliminary results]. Rev Laryngol Otol Rhinol 2009;130:175–180.

7. Jang ES, Park JW, Kweon H, Lee KG, Kang SW, Baek DH, Choi

JY, Kim SG. Restoration of peri-implant defects in immediate

implant installations by Choukroun platelet-rich fibrin and silk

fibroin powder combination graft. Oral Surg Oral Med Oral Pathol

Oral Radiol Endod 2010;109:831–836.

8. Dohan DM, Choukroun J, Diss A, Dohan SL, Dohan AJ, Mouhyi J,

Gogly B. Platelet-rich fibrin (PRF): A second-generation platelet

concentrate. Part I: Technological concepts and evolution. Oral

Surg Oral Med Oral Pathol Oral Radiol Endod 2006;101:e37–e44.

9. Dohan Ehrenfest DM, de Peppo GM, Doglioli P, Sammartino G. Slow

release of growth factors and thrombospondin-1 in Choukroun’s pla-

telet-rich fibrin (PRF): A gold standard to achieve for all surgical plate-

let concentrates technologies. Growth Factors 2009;27:63–69.

10. He L, Lin Y, Hu X, Zhang Y, Wu H. A comparative study of plate-

let-rich fibrin (PRF) and platelet-rich plasma (PRP) on the effect of

proliferation and differentiation of rat osteoblasts in vitro. Oral

Surg Oral Med Oral Pathol Oral Radiol Endod 2009;108:707–713.

11. Choukroun JI, Braccini F, Diss A, Giordano G, Doglioli P, Dohan

DM. [Influence of platelet rich fibrin (PRF) on proliferation of

human preadipocytes and tympanic keratinocytes: A new oppor-

tunity in facial lipostructure (Coleman’s technique) and tympano-

plasty?]. Rev Laryngol Otol Rhinol 2007;128:27–32.

12. Dohan Ehrenfest DM, Diss A, Odin G, Doglioli P, Hippolyte MP,

Charrier JB. In vitro effects of Choukroun’s PRF (platelet-rich fibrin)

on human gingival fibroblasts, dermal prekeratinocytes, preadipo-

cytes, and maxillofacial osteoblasts in primary cultures. Oral Surg

Oral Med Oral Pathol Oral Radiol Endod 2009;108:341–352.

13. Dohan Ehrenfest DM, Doglioli P, de Peppo GM, Del Corso M,

Charrier JB. Choukroun’s platelet-rich fibrin (PRF) stimulates in

vitro proliferation and differentiation of human oral bone mesen-

chymal stem cell in a dose-dependent way. Arch Oral Biol 2010;

55:185–194.

14. Rowlands AS, George PA, Cooper-White JJ. Directing osteogenic

and myogenic differentiation of MSCs: Interplay of stiffness and

adhesive ligand presentation. Am J Physiol Cell Physiol 2008;295:

C1037–C1044.

15. Evans ND, Minelli C, Gentleman E, LaPointe V, Patankar SN, Kal-

livretaki M, Chen X, Roberts CJ, Stevens MM. Substrate stiffness

affects early differentiation events in embryonic stem cells. Eur

Cell Mater 2009;18:1–13; discussion 13–14.

16. Boyan BD, Lossdorfer S, Wang L, Zhao G, Lohmann CH, Cochran

DL, Schwartz Z. Osteoblasts generate an osteogenic microenvir-

onment when grown on surfaces with rough microtopographies.

Eur Cell Mater 2003;6:22–27.

ORIGINAL ARTICLE

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2012 VOL 00A, ISSUE 00 11

17. Le Nihouannen D, Saffarzadeh A, Gauthier O, Moreau F, Pilet P,

Spaethe R, Layrolle P, Daculsi G. Bone tissue formation in sheep

muscles induced by a biphasic calcium phosphate ceramic and

fibrin glue composite. J Mater Sci Mater Med 2008;19:667–675.

18. Filmon R, Basle MF, Atmani H, Chappard D. Adherence of osteo-

blast-like cells on calcospherites developed on a biomaterial com-

bining poly(2-hydroxyethyl) methacrylate and alkaline phosphatase.

Bone 2002;30:152–158.

19. Filmon R, Basle MF, Barbier A, Chappard D. Poly(2-hydroxy ethyl

methacrylate)-alkaline phosphatase: A composite biomaterial

allowing in vitro studies of bisphosphonates on the mineralization

process. J Biomater Sci Polym Ed 2000;11:849–868.

20. Filmon R, Basle M. F, Barbier A, Chappard D. [In vitro study of

the effect of bisphosphonates on mineralization induced by a

composite material: Poly 2(hydroxyethyl) methacrylate coupled

with alkaline phosphatase]. Morphologie 2000;84:3–33.

21. Beertsen W, van den Bos T. Alkaline phosphatase induces the

mineralization of sheets of collagen implanted subcutaneously in

the rat. J Clin Invest 1992;89:1974–1980.

22. Yamauchi K, Goda T, Takeuchi N, Einaga H, Tanabe T. Prepara-

tion of collagen/calcium phosphate multilayer sheet using enzy-

matic mineralization. Biomaterials 2004;25:5481–5489.

23. Spoerke ED, Anthony SG, Stupp SI. Enzyme directed templating

of artificial bone mineral. Adv Mater 2009;21:425–430.

24. Bruder SP, Caplan AI. A monoclonal antibody against the surface

of osteoblasts recognizes alkaline phosphatase isoenzymes in

bone, liver, kidney, and intestine. Bone 1990;11:133–139.

25. Kang YH, Jeon SH, Park JY, Chung JH, Choung YH, Choung HW,

Kim ES, Choung PH. Platelet-rich fibrin is a Bioscaffold and reser-

voir of growth factors for tissue regeneration. Tissue Eng Part A

2010;17:349–359.

26. Antoniades HN. Human platelet-derived growth factor (PDGF): Pu-

rification of PDGF-I and PDGF-II and separation of their reduced

subunits. Proc Natl Acad Sci USA 1981;78:7314–7317.

27. Tischer E, Gospodarowicz D, Mitchell R, Silva M, Schilling J, Lau

K, Crisp T, Fiddes JC, Abraham JA. Vascular endothelial growth

factor: A new member of the platelet-derived growth factor gene

family. Biochem Biophys Res Commun 1989;165:1198–1206.

28. Pfeilschifter J, Bonewald L, Mundy GR. Characterization of the

latent transforming growth factor beta complex in bone. J Bone

Miner Res 1990;5:49–58.

29. Gassling V, Douglas T, Warnke PH, Acil Y, Wiltfang J, Becker ST.

Platelet-rich fibrin membranes as scaffolds for periosteal tissue

engineering. Clin Oral Implants Res 2010;21:543–549.

30. Gassling VL, Acil Y, Springer IN, Hubert N, Wiltfang J. Platelet-

rich plasma and platelet-rich fibrin in human cell culture. Oral

Surg Oral Med Oral Pathol Oral Radiol Endod 2009;108:48–55.

31. Schouten C, van den Beucken JJ, de Jonge LT, Bronkhorst EM,

Meijer GJ, Spauwen PH, Jansen JA. The effect of alkaline phos-

phatase coated onto titanium alloys on bone responses in rats.

Biomaterials 2009;30:6407–6417.

32. Brandl FP, Seitz AK, Tessmar JK, Blunk T, G€opferich AM. Enzy-

matically degradable poly(ethylene glycol) based hydrogels for

adipose tissue engineering. Biomaterials 2010;31:3957–3966.

33. Park H, Guo X, Temenoff JS, Tabata Y, Caplan AI, Kasper FK,

Mikos AG. Effect of swelling ratio of injectable hydrogel compo-

sites on chondrogenic differentiation of encapsulated rabiit marrow

mesenchymal stem cells. Biomacromolecules 2009;10:541–546.

34. Douglas T, Liu Q, Humpe A, Wiltfang J, Sivananthan S, Warnke

PH. Novel ceramic bone replacement material CeraBall seeded

with human mesenchymal stem cells. Clin Oral Implants Res

2010;21:262–267.

35. Douglas T, Pamula E, Hauk D, Wiltfang J, Sivananthan S, Sherry

E, Warnke PH. Porous polymer/hydroxyapatite scaffolds: Charac-

terization and biocompatibility investigations. J Mater Sci Mater

Med 2009;20:1909–1915.

36. Warnke PH, Douglas T, Sivananthan S, Wiltfang J, Springer I,

Becker ST. Tissue engineering of periosteal cell membranes in

vitro. Clin Oral Implants Res 2009;20:761–766.

37. Warnke PH, Douglas T, Wollny P, Sherry E, Steiner M, Galonska

S, Becker ST, Springer IN, Wiltfang J, Sivananthan S. Rapid pro-

totyping: Porous titanium alloy scaffolds produced by selective

laser melting for bone tissue engineering. Tissue Eng Part C

Methods 2009;15:115–124.

38. Koutsopoulos S. Synthesis and characterization of hydroxyapatite

crystals: A review study on the analytical methods. J Biomed

Mater Res 2002;62:600–612.

39. Orimo H. The mechanism of mineralization and the role of alka-

line phosphatase in health and disease. J Nippon Med Sch 2010;

77:4–12.

40. de Jonge LT, van den Beucken JJ, Leeuwenburgh SC, Hamers

AA, Wolke JG, Jansen JA. In vitro responses to electrosprayed

alkaline phosphatase/calcium phosphate composite coatings. Acta

Biomater 2009;5:2773–2782.

41. Berendsen AD, Smit TH, Hoeben KA, Walboomers XF, Bronckers AL,

Everts V. Alkaline phosphatase-induced mineral deposition to anchor

collagen fibrils to a solid surface. Biomaterials 2007;28:3530–3536.

42. Osathanon T, Giachelli CM, Somerman MJ. Immobilization of

alkaline phosphatase on microporous nanofibrous fibrin scaffolds

for bone tissue engineering. Biomaterials 2009;30:4513–4521.

43. Le Guehennec L, Goyenvalle E, Aguado E, Pilet P, Bagot D’Arc M,

Bilban M, Spaethe R, Daculsi G. MBCP biphasic calcium phos-

phate granules and tissucol fibrin sealant in rabbit femoral

defects: The effect of fibrin on bone ingrowth. J Mater Sci Mater

Med 2005;16:29–35.

44. Le Guehennec L, Goyenvalle E, Aguado E, Pilet P, Spaethe R,

Daculsi G. Influence of calcium chloride and aprotinin in the in

vivo biological performance of a composite combining biphasic

calcium phosphate granules and fibrin sealant. J Mater Sci Mater

Med 2007;18:1489–1495.

45. Le Guehennec L, Layrolle P, Daculsi G. A review of bioceramics

and fibrin sealant. Eur Cell Mater 2004;8:1–10; discussion 10–11.

46. Jegoux F, Goyenvalle E, Bagot D’arc M, Aguado E, Daculsi G. In

vivo biological performance of composites combining micro-mac-

roporous biphasic calcium phosphate granules and fibrin sealant.

Arch Orthop Trauma Surg 2005;125:153–159.

47. Saffarzadeh A, Gauthier O, Bilban M, Bagot D’Arc M, Daculsi G.

Comparison of two bone substitute biomaterials consisting of a

mixture of fibrin sealant (Tisseel) and MBCP (TricOs) with an

autograft in sinus lift surgery in sheep. Clin Oral Implants Res

2009;20:1133–1139.

48. Le Nihouannen D, Guehennec LL, Rouillon T, Pilet P, Bilban M,

Layrolle P, Daculsi G. Micro-architecture of calcium phosphate

granules and fibrin glue composites for bone tissue engineering.

Biomaterials 2006;27:2716–2722.

49. Banks E, Nakajima S, Shapiro LC, Tilevitz O. Fibrous apatite on

modified collagen. Science 1997;198:1164–1166.

50. Doi Y, Horiguchi T, Moriwaki Y, Kitago H, Kajimoto T, Iwayama Y.

Formation of apatite-collagen complexes. J Biomed Mater Res

1996;31:43–49.

51. Feng CF, Khor KA, Kwek SWK, Cheang P. Thermally induced crys-

tallization of amorphous calcium phosphate in plasma-spheroi-

dised hydroxyapatite powders. Mater Lett 2000;46:229–233.

52. Van den Vreken NM, Pieters IY, Declercq HA, Cornelissen MJ,

Verbeeck RM. Characterization of calcium phosphate cements

modified by addition of amorphous calcium phosphate. Acta Bio-

mater 2010;6:617–625.

53. Durand B, Gao FB, Raff M. Accumulation of the cyclin-dependent

kinase inhibitor p27/Kip1 and the timing of oligodendrocyte differ-

entiation. EMBO J 1997;6:306–317.

54. Anh DJ, Eden A, Farley JR. Quantitation of soluble and skeletal

alkaline phosphatase, and insoluble alkaline phosphatase anchor-

hydrolase activities in human serum. Clin Chim Acta 2001;311:

137–148.

55. Valenzuela GJ, Munson LA, Tarbaux NM, Farley JR. Time-depend-

ent changes in bone, placental, intestinal, and hepatic alkaline

phosphatase activities in serum during human pregnancy. Clin

Chem 1987;33:1801–1806.

56. Su CY, Kuo YP, Tseng YH, Su CH, Burnouf T. In vitro release of

growth factors from platelet-rich fibrin (PRF): A proposal to opti-

mize the clinical applications of PRF. Oral Surg Oral Med Oral

Pathol Oral Radiol Endod 2009;108:56–61.

12 DOUGLAS ET AL. ENZYMATICALLY INDUCED MINERALIZATION OF PRF