LNGFR Induction During Osteogenesis of Human Jaw Periosteum-derived Cells

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Original Paper

Cell Physiol Biochem 2009;24:283-290 Accepted: June 02, 2009Cellular PhysiologyCellular PhysiologyCellular PhysiologyCellular PhysiologyCellular Physiologyand Biochemistrand Biochemistrand Biochemistrand Biochemistrand Biochemistryyyyy

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LNGFR Induction During Osteogenesis of HumanJaw Periosteum-derived Cells

Dorothea Alexander1, Fabian Schäfer1, Adelheid Munz1, BjörnFriedrich2, Christian Klein3, Jürgen Hoffmann1, Hans-Jörg Bühring4

and Siegmar Reinert1

1Department of Oral and Maxillofacial Surgery, University Hospital Tübingen, 2Department of InternalMedicine IV, University Hospital Tübingen, 3Department of Conservative Dentistry, University HospitalTübingen, 4Department of Internal Medicine II, University Hospital Tübingen

Dr. Dorothea Alexander, PhDOral and Maxillofacial Surgery, University Hospital TübingenOsianderstr. 2-8, 72076 Tübingen (Germany)Tel. +49-7071-2982418, Fax +49-7071-294365E-Mail [email protected]

Key WordsLNGFR • Osteogenic capacity • Jaw periosteal cells •Mineralization • Differentiation marker

AbstractIsolated jaw periosteum-derived cells (JPCs) comprisea morphologically heterogeneous population. Thereare no known specific surface markers that are ableto distinguish between progenitors and cells of othertissue types. The aim of our study was to identifydifferentiation markers as predictors of JPCmineralization capacity. JPCs underwent osteogenicdifferentiation after cultivation in osteogenic mediumcontaining known activators. By FACS analysis, wefound the low affinity nerve growth factor receptor(LNGFR–CD271) to be induced during the first fivedays of osteogenesis and that it was expressed athigher levels in mineralizing JPCs (mJPCs) incomparison to non-mineralizing JPCs (nmJPCs).Similar results were obtained by semi-quantitativeimmunohistochemical stainings and western blotanalyses. Quantitative real-time PCR results showedsignificantly higher LNGFR and alkaline phosphatasetranscript levels in mJPCs compared to nmJPCs.LNGFR is a differentiation marker that distinguishes

between mineralizing JPCs and non-mineralizing JPCsduring the first phase of osteogenesis and cantherefore be considered an early surface marker ofosteogenic capacity in vitro.

Introduction

For bone tissue engineering applications differentmesenchymal stem cell sources could be used, howeverdepending on their localization they show differentexpression profiles [1-3]. Despite the fact that jawperiosteum-derived cells (JPCs) have some advantagesover bone marrow-derived mesenchymal stem cells(BMMSCs) for applications in oral and maxillofacialsurgery [1], they are still poorly characterized and little isknown about their antigenic profile and differentiationbehaviour. JPCs have a specialized function within thejaw and their localization commonly overlaps with thesite of oral or maxillofacial surgery. The osteogenicpotential of tibial periosteum-derived cells remains thesame as age increases [4]. This is not the case forBMMSCs, which are known to lose their osteogenicpotential with increases in age [5]. The osteogenicpotential and bone capacity of BMMSCs in comparison

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to isogenic JPCs was already investigated [2]. The resultsshowed a delayed and overall reduced capacity of JPCsto differentiate toward the osteoblastic lineage ascompared to BMMSCs [2]. However, a recently publishedwork demonstrated an effective tissue engineering usingperiosteal cells [1].

The low-affinity nerve growth factor receptor(LNGFR), also referred to as the p75 neutrophin receptor(p75NTR), tumor necrosis factor receptor superfamilymember 16 (TNFRSF16) or CD271, was described asthe most selective marker for the purification andphenotypic characterization of BMMSC [6]. LNGFR, isa transmembrane protein capable of binding nerve growthfactor [7]. Whereas LNGFR binds to the precursors ofneurotrophic factors with low affinity, it is required forefficient Trk kinase activation and signaling [7]. LNGFRalso has been proven to be an useful marker for theisolation of adipose tissue-derived stem cells in a mouseanimal model [8].

After JPC isolation, a heterogeneous cell populationcontaining several cell types is obtained. We found twocell groups that act differently during osteogenesis: onecell group is not able to mineralize in vitro (non-mineralizingJPCs-nmJPCs), and the other cell group is able tomineralize in vitro (mineralizing JPCs-mJPCs). As specificmarkers that distinguish between progenitor and othercell types are currently lacking, we aimed to identifypredictors of JPC osteogenic capacity in order to improvethe quality of this stem cell type for bone regeneration.

In the present study, we detected a significantelevation of the cell surface marker LNGFR (CD271) inosteogenic differentiated JPCs during the first five daysof osteogenesis.

Materials and Methods

Cell culturesBiopsies of human jaw periosteum were obtained during

routine maxillofacial interventions. Samples from 14 donors wereincluded in the study in accordance with the local ethicalcommittee, following informed consent. JPCs were isolated in apredigestion step by type XI collagenase (1500 U/ml, Sigma,Steinheim, Germany) and dispase II (2.4 U/ml, Roche AppliedSciences, Mannheim, Germany) in a 1:1 mixture for 30 min. Themain digestion step followed for 90 min with type XI collagenase.

JPCs were cultured in D-MEM: F-12 (Invitrogen-BioSource Europe, Nivelles, Belgium) containing 10% FCS(Sigma-Aldrich, Steinheim, Germany), 100 U/ml penicillin/streptomycin (Cambrex, Walkersville, USA) and 50 µg/mlfungicide (Biochrom AG, Berlin, Germany). Periosteal cells fromthe third to sixth passages were used for these experiments.

After isolation of quite a few patient JPCs, we alwaysobtained a mixed and heterogenous cell population containingdifferent cell types (tissue fibroblasts and others) and probablydifferent stages of possible progenitor cells. A reliableseparation of the outer (fibrous tissue) from the inner layer(cambium layer containing the progenitor cells) of periosteumwas not possible. On the basis of our results and with respectto their osteogenic potential, we classified the isolated andexpanded cells afterwards into two groups: 1) non-mineralizingJPCs (nmJPCs), which were not able to calcify in vitro (n=7)and 2) mineralizing JPCs (mJPCs), which were able to calcify invitro (n=7). JPC mineralization was detected by alizarin staining.nmJPCs showed an alizarin-negative staining after 20 days ofosteogenesis (percentage of mineralization 0%). mJPCs clearlyshowed an alizarin-positive (reddish dye) staining after thesame period of osteogenic differentiation (percentage ofmineralization 50-90%). Cell cultures with lower mineralizationpotential were not included in the study. However, these twocell culture types were derived from different and not the samedonors. We did not detect clear morphological differencesbetween nmJPCs and mJPCs, which would make a simplemorphological differentiation between both cell typespracticable.

Differentiation experimentsFor the osteogenic differentiation experiments, mJPCs and

nmJPCs were cultured in D-MEM/10% FCS, 4 µMdexamethasone, 10 mM β-glycerolphosphate and 100 µM L-ascorbic acid 2-phosphate (Sigma-Aldrich, Germany). Cells weretreated with these components for a period of 20 days in theabsence (referred to as OB-medium) or presence of 100 ng/mlhuman recombinant BMP-2 (referred to as OB/BMP-2). Theculture medium was replaced every two days. As controls, weused untreated JPCs that were cultivated in the absence of anyosteogenic components for the same time period.

Alizarin and von Kossa stainingCalcium deposition of JPCs was detected by alizarin

staining. Therefore, cells were fixed in ice-cold methanol for 2min, air-dried and stained with alizarin for 20 min. Differentiatedcells containing mineral deposits were stained bright red. TheJPC mineralization capacity was also detected by an alternativemethod, von Kossa silver staining. Fixed cells were incubatedwith 5% silver nitrate solution for one hour in the dark. Reductionwith sodium carbonate and fixation with 5% sodium thiosulfatefollowed for 2 min.

RNA extraction and quantitative RT-PCRTotal cellular RNA was extracted from differentiated cells

and untreated controls using the RNeasy Mini Kit (Qiagen,Hilden, Germany) as recommended by the manufacturer. First-strand cDNA for the real-time PCR experiments was synthesizedby reverse transcription at 42°C in a 20 µl reaction mixturecontaining 1 µg of total RNA. After heating the samples at95°C for 10 min for denaturation and cooling to 4°C, cDNA wasused for PCR amplification. Reactions were diluted to 100 µlwith DEPC-treated water.

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To quantify mRNA expression, real-time PCR with theLightCycler System (Roche Diagnostics, Mannheim, Germany)was performed (for detail [9]). PCRs for all analysed genes wereperformed in 20 µl final volumes of containing 2 µl cDNA, 2 µlprimer mix (commercial primer kits from Search LC, Heidelberg,Germany), 2 µl DNA Master Sybr Green I mix (Roche MolecularBiochemicals, Mannheim, Germany) and 14 µl DEPC-treatedwater. Amplification of the target DNA was performed during35-38 cycles of 95°C for 10 s, 68°C for 10 s and 72°C for 16 s,each with a temperature transition rate of 20°C/s and asecondary target temperature of 58°C with a step size of 0.5°C.Melting curve analysis was performed at 95°C 0 s, 58°C 10 s,95°C 0 s to allow discrimination between unspecific primer dimersand specific PCR products. The transcript levels of thehousekeeping gene GAPDH and alkaline phosphatase (AP),osterix (Osx), Runx-2 (Cbfa1) and insulin-like growth factor-2(IGF-2) were also determined for each sample using a commercialprimer kit (Search LC, Heidelberg, Germany). Finally, resultswere calculated as a ratio of the target versus the housekeepinggene transcripts.

Analysis of LNGFR protein expressionLNGFR immunohistochemistry was performed after

fixation of cells in ice-cold methanol for 5 min. After a hydrogenperoxide block for 7 min and an additional wash step with PBS,cells were incubated with the monoclonal mouse antibody anti-LNGFR (ME20.4, 1:500, Santa Cruz Biotechnology, USA) for1.5 hours at room temperature. For the following steps, theNOVA Detect HRPO/DAB Quick Kit (Dianova, Germany) wasused.

For western blot analysis pelleted cells were dissolved in60 µl lysis buffer (1% NP40; 150 mM NaCl; 50 mM Tris/HCL,pH 8.0 plus protease inhibitors) which were stored overnight

at -70°C. Cell debris were removed by centrifugation and proteinconcentrations of the supernatants were measured using acolorimetric assay (RD DC Protein Assay, BioRad, Germany).After addition of reducing Laemmli sample buffer, a total of 50µg of nondenaturated cell lysate from the cellular extracts wasseparated by SDS-PAGE (10%) and transferred ontonitrocellulose membranes. The membranes were analyzed byimmunoblotting using monoclonal mouse anti-LNGFR (ME20.41:1000, Santa Cruz, USA) and anti-GAPDH (Abcam, UK) specificprimary antibodies overnight at 4°C. Binding of the primaryantibodies was detected with peroxidase-conjugated goat anti-mouse secondary antibodies (Santa Cruz, USA) and visualizedby the enhanced chemiluminescence method (GE Healthcare,Germany).

Flow cytometry analysis of LNGFRAfter cell detachment with 0.5% trypsin and washing with

PBS, cell pellets were incubated with 60 µl GAMUNEX 10%(human immune globulin solution) for 15 min at 4°C to blockunspecific labelling. After an additional wash step, up to 104

cells were incubated with 10 µl mouse monoclonal antibodiesconjugated to allophycocyanin (CD271-APC, mouse IgG1,clone ME20.4-1H4, Miltenyi Biotec, Germany) for 15 min at4°C. Cells were washed twice with FACS buffer (PBS/1% BSA/0.01% NaN3) and analysed on a FACSCanto flow cytometer(Becton Dickinson, Germany). Data were processed using theFCS Express software (DeNovo, Canada).

Statistical analysisData are expressed as means ± SEM. Statistical analysis

was performed using non-parametric paired t-tests for data witha normal distribution. A value of p< 0.05 was consideredsignificant.

LNGFR Induction During JPC Osteogenesis

Fig. 1. Osteogenic potential of jawperiosteal cells. Periosteal cells underwentosteogenesis (OB=osteoblast specificmedium, OB/BMP-2=osteoblast specificmedium and BMP-2) and mineralizationpotential was tested after 20 days ofdifferentiation by the alizarin (upper panel)and von Kossa (lower panel) staining(magnification 4x) in comparison to untreatedcells (co=control cells). We found two cellgroups: mineralizing JPCs (mJPCs) and non-mineralizing JPCs (nmJPCs). Detectedcalcium-phosphate precipitates formed fromJPCs, resulting in a reddish staining afteraddition of alizarin and in a black stainingafter silver staining. Negative staining wasobserved in untreated and OB/OB-BMP-2nmJPCs.

Cell Physiol Biochem 2009;24:283-290

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Results

Osteogenic potential of JPCsThe mineralization capacity of differentiating JPCs

was determined after 20 days of culture using alizarinand von Kossa stainings. We classified the isolated andexpanded cells into two groups: non-mineralizing JPCs

Fig. 2. AP and LNGFR gene expression in mJPCs vs. nmJPCs. AP (Fig. 2A) and LNGFR (Fig. 2B) gene expression was quantifiedin mJPCs/nmJPCs (n=7 for each group) at days 5, 10 and 20 of osteogenesis by real-time PCR. Ratios of AP or LNGFR/GAPDH aredenoted in the y-axis. * p<0.05. Significantly elevated AP transcript levels were detected in mJPCs versus nmJPCs at all analysedtime points (days 5, 10, 20) in both untreated controls and differentiating cells. Significantly elevated LNGFR transcript levelswere found in OB-treated mJPCs vs. nmJPCs. The same tendency of higher LNGFR gene expression was observed in OB/BMP-2-treated mJPCs vs. nmJPCs, however, the differences achieved no significant values.

Day 5

B

(nmJPCs, n=7), which were not able to calcify in vitroand mineralizing JPCs (mJPCs, n=7), which developedstrong reddish staining with containing calcium-phosphateprecipitates (Fig. 1 upper panel). Similarly, von Kossasilver staining of nmJPCs resulted in a negative reaction,whereas calcium-precipitates formed in mineralizing JPCs,resulting in a black staining (Fig. 1, lower panel).

Alexander/Schäfer/Munz/Friedrich/Klein/Hoffmann/Bühring/Reinert

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Day 10

Day 20

Induction of AP gene expression inmJPC versus nmJPC

Induction of LNGFR gene expressionin mJPC versus nmJPC

Day 5

Day 10

Day 20


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Gene expression analyses of AP and LNGFR inmJPCs versus nmJPCsExpression levels of alkaline phosphatase were

significantly elevated in mJPCs versus nmJPCs in bothuntreated and osteogenic differentiating cells (Fig. 2A).

LNGFR Induction During JPC Osteogenesis

nmJPCs also responded to the osteogenic stimuli with aninduction of AP gene expression, however, the levels weresignificant lower than those detected in mJPCs. At day5, we found higher AP/GAPDH ratios in untreatedmJPCs (13.87±3.41) compared to untreated nmJPCs

A

B

Fig. 3. LNGFR protein expression inmJPCs vs. nmJPCs. mJPCs/nmJPCsunderwent osteogenesis and LNGFRprotein expression was detected afterday 20 by immunohistochemistry (Fig.3A, magnification 4x) or by Westernblotting (Fig. 3B). As a positivecontrol, whole cell lysates from humanneuroepithelioma cells were used. Asloading controls, GAPDH proteinexpression was detected in the samelysates. Representative stainings/blots for mJPCs and nmJPCs areillustrated.

Fig. 4. FACS analysis of LNGFR inmJPCs vs. nmJPCs. For immuno-fluorescence staining, untreated anddifferentiating JPCs were incubatedwith allophycocyanin-conjugatedmonoclonal LNGFR antibodies andanalysed on a FACSCanto flowcytometer. Representativehistograms for LNGFR expression areillustrated in Fig. 4A. Average valuesof percent LNGFR positive cells (n=6for mJPC and nmJPC) are indicated inthe histograms. MFI (medianfluorescence intensity) data areshown in Fig. 4B *. p<0.05.

A

B


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(2.85±0.83). This difference was highly significant(p<0.01) and remained almost the same at day 10 andday 20. Five days after initiation of osteogenesis, wedetected in OB- and OB/BMP-2 treated mJPCs stronglyelevated AP/GAPDH ratios (53.57±12.05 and53.44±10.79, respectively), which were significantlyhigher than those in OB- and OB/BMP-2 treated nmJPCs(5.96±2.2 and 7.17±1.24, respectively). Likewise, wefound at day 10 and day 20 of differentiation significantlyhigher AP levels in OB- and OB/BMP-2 treated mJPCs.

LNGFR gene expression was analyzed byquantitative real-time PCR in mJPCs versus nmJPCs atdays 5, 10 and 20 of osteogenesis (Fig. 2B). At allanalyzed time points, OB- and OB/BMP-2 treated mJPCs(n=7) showed higher LNGFR transcript levels thannmJPCs (n=7, Fig. 2B). At the first analyzed time point(day 5), transcript levels of LNGFR were slightly, butsignificantly increased in untreated controls from mJPCsversus nmJPCs (mJPCs: 0.23±0.05; nmJPCs 0.09±0.02).Induction of osteogenesis led to higher LNGFR/GAPDHratios by day 5 in OB- and OB/BMP-2 treated mJPCs(day 5: 4.46±1.41 and 4.82±2.17) compared to treatednmJPCs (day 5: 0.28±0.1 and 0.38±0.18, respectively).The same tendency was found at days 10 and 20 ofdifferentiation in OB- and OB/BMP-2 treated mJPCs(day 10: 7.7±3.2 and 5.05±2.51; day 20: 8.21±2.55 and12.59±5.57) versus OB- and OB/BMP-2 treated nmJPCs(day 10: 0.4±0.18 and 0.41±0.13; day 20: 1.0±0.4 and0.74±0.22). However, differences in LNGFR geneexpression between mJPCs and nmJPCs achievedsignificant values only in OB-treated cells.

LNGFR protein expression in mJPCs versusnmJPCsLNGFR protein expression was analyzed at the same

time points as chosen for quantitative PCR analysis (days5, 10 and 20 of osteogenesis). After initiation ofosteogenesis in JPCs, protein levels of LNGFR weredetected by immunohistochemistry. A slight induction ofLNGFR protein expression was already observed at thebeginning of differentiation (day 5, data not shown), whichgradually increased through the end of osteogenesis inmJPCs at day 20 (Fig. 3A).

Similar to the quantitative PCR results, an inductionof LNGFR expression was also detected by western blotwith gradually increasing intensity during osteogenesis inOB- and OB/BMP-2 treated mJPCs (Fig. 3B). Whenresults from mJPCs were compared with those fromnmJPCs, much stronger signals could be detected in themJPCs group.

Flow cytometry analysis of LNGFR expressionin mJPCs versus nmJPCsLNGFR expression on cell surface was detected

by FACS analysis at the beginning of osteogenesis.Representative histograms for LNGFR expression inmJPCs (n=6) versus nmJPCs (n=6) after day 5 ofosteogenesis are illustrated in Fig. 4A. In untreated mJPCswe found on average 44% LNGFR positive cells, whereasnmJPCs were shown to be on average 60% positive forLNGFR. However, FACS analysis showed a particularpattern of LNGFR expression in osteogenic differentiatedJPCs. Due to OB or OB/BMP-2 treatment of mJPCs,cells showed a LNGFRbright expression which reflects aLNGFR induction (on average 62%) during osteogenesis.OB or OB/BMP-2 treated nmJPCs revealed a LNGFRdim

expression which indicates a repression of LNGFRpresentation on cell surface (on average 49% and 45%respective).

Analysis of the median fluorescence intensityrevealed significantly higher levels of LNGFR expressionin OB-treated mJPCs than in OB-treated nmJPCs afterinitiation of osteogenesis (day 5: OB-treated mJPCs1.81±0.28 versus OB-treated nmJPCs 0.86±0.03, p<0.01,Fig. 4B). The same tendency was observed in OB/BMP-2-treated cells, however, the differences were notconsidered significant.

Discussion

Despite the fact that isolated jaw periosteum-derivedcells (JPCs) could be used for bone tissue applications,these cells represent a morphologically heterogeneouspopulation. Furthermore, due to the unavailability ofspecific markers for JPCs, we run the risk of other celltypes outgrowing progenitor cells, resulting in the culturingof a cell population that lacks progenitor cells. Ourobservation that not all isolated JPCs are able to mineralizein vitro could be due to the concomitance of different celltypes, which might interfere with the complexmineralization process. For isolation and characterizationof bone marrow-derived mesenchymal stem cells(BMMSCs) [10] and of adipose tissue-derived stem cells[8] the low-affinity nerve growth factor receptor(LNGFR) is one of the key markers. In previous studiesthe expression of LNGFR and alkaline phosphatase (AP)was almost undetectable in undifferentiated tibial periostealcells [11]. These results are in line with our data inundifferentiated control JPCs. In the present work, weidentified LNGFR transcripts to be differentially modulated


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within the first five days of JPCs osteogenic differentiationprocess. Furthermore, western blot data demonstratehigher protein levels of LNGFR in mineralizing JPCs(mJPCs) compared to non-mineralizing JPCs (nmJPCs)at this early time point. A similar pattern on transcript andprotein levels can be found at day 10 and day 20. Theseresults indicate a role for LNGFR as an earlier markerfor JPC mineralization capacity than the Alizarin or vonKossa stainings, which are useful only after 20 days ofdifferentiation. LNGFR is a surface marker which couldbe used for isolation procedures. In general, by FACSanalysis LNGFR showed nearly an opposite pattern ofexpression in OB- and OB/BMP-2 treated mJPCs incontrast to identical treated nmJPCs. Other groups hasbeen investigated LNGFR in different settings. Recently,LNGFR positive cells were isolated from the growth zonesof regenerating fallow deer antlers as well as the pedicleperiosteum [12]. The alteration of LNGFR expressionfollowing differentiation has been investigated also inmesenchymal stem cells [13]. This group differentiatedMSCs along a Schwann cell lineage and showed thatLNGFR expression was strongly elevated upondifferentiation [13].

LNGFR was shown to modulate the expression ofgenes that are critically involved in the regulation ofdifferentiation as well as cell adhesion, signal transduction,apoptosis, tumor cell invasion, and metastasis [14].Numerous adaptor proteins that bind LNGFR have beenreported [15]. Increased LNGFR levels were found tobe associated with reduced levels of MMP-9 andincreased levels of TIMP-1 [16]. Re-expression of lost

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LNGFR Induction During Osteogenesis of Human Jaw Periosteum-derived Cells

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