research article potential osteoinductive effects of

Research ArticlePotential Osteoinductive Effects of Calcitriol onthe m-RNA of Mesenchymal Stem Cells Derived fromHuman Alveolar Periosteum

Hsiang-Hsi Hong123 Adrienne Hong4 Tzung-Hai Yen2567 and Yen-Li Wang28

1Department of Periodontics Chang Gung Memorial Hospital Linkou Taiwan2Chang Gung University Taoyuan Taiwan3School of Dental Technology College of Oral Medicine Taipei Medical University Taipei Taiwan4California Northstate University College of Medicine Elk Grove CA USA5Division of Clinical Toxicology Department of Nephrology Chang Gung Memorial Hospital Linkou Taiwan6Kidney Research Center Chang Gung Memorial Hospital Linkou Taiwan7Center for Tissue Engineering Chang Gung Memorial Hospital Linkou Taiwan8Department of Periodontics Chang Gung Memorial Hospital Taoyuan Taiwan

Correspondence should be addressed to Yen-Li Wang wanglacy519yahoocomtw

Received 24 June 2016 Revised 18 October 2016 Accepted 30 October 2016

Academic Editor Kunikazu Tsuji

Copyright copy 2016 Hsiang-Hsi Hong et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

This study characterized alveolar periosteum-derived mesenchymal stem cells (P-MSCs) and examined the hypothesis that 125-(OH)2D3 (calcitriol) exerts osteoinductive effects on P-MSCs The mRNA expressions of alkaline phosphatase (ALP) bonesialoprotein (BSP) core-binding factor alpha-1 (CBFA1) collagen-1 (Col-1) osteocalcin (OCN) and vitamin D3 receptor (VDR)were assessed after incubation with calcitriol for 2 weeks Vitamin C as positive control (Vit C-p) increased ALP and CBFA1mRNAexpression at both 1 and 2 weeks and increased BSP and Col-1 mRNA expression only at the first week A concentration of 10minus8Mcalcitriol enhanced ALP CBFA1 Col-1 and OCN mRNA expression at both weeks and BSP mRNA expression at the first weekFurthermore 10minus7M calcitriol increased the mRNA expressions of all compounds at both weeks except that of CBFA1 at the firstweek 10minus8M calcitriol and Vit C-p enhanced ALP activity at the second and third weeks The results revealed that 10minus9 10minus8 and10minus7M calcitriol induced osteoinduction in alveolar P-MSCs by increasing ALP CBFA1 Col-1 and OCN mRNA expression A10minus7M calcitriol yielded a higher mRNA expression than Vit Cp on VDR and OCNmRNA expression at both weeks and on Col-1mRNA at the second week

1 Introduction

Mesenchymal stem cells (MSCs) can be isolated from varioustissues including long bone periosteumand dental tissues [1]To date six types of human dental stem cells namely stemcells from postnatal dental pulp [2] exfoliated deciduousteeth [3] periodontal ligament [4] apical papilla [5] dentalfollicle [6] and gingival tissue [7] have been isolated fromand characterized with the parent tissuesThe cambium layerof the periosteum primarily contains a mixed cell populationof fibroblasts [8] osteoblasts [9] and pericytes [10] and a crit-ical subpopulation identified as MSCs [11] The accessibility

of the periosteum and favorable bone regeneration resultshave been reported [12] and an increasing number of dentistsare applying alveolar bone regeneration in periodontologyand implant dentistry However few studies have exploredthe regenerative potential of alveolar periosteum MSCs (P-MSCs) in dentistry

Vitamin D a group of fat-soluble secosteroids is crucialfor enhancing the intestinal absorption of calcium ironmagnesium phosphate and zinc Calcitriol [125-(OH)2D3Vit D3] circulates as a hormone in the blood adjusts calciumand phosphate concentrations in the circulatory system andsupports remodeling and healthy bone growth Furthermore

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 3529561 10 pageshttpdxdoiorg10115520163529561

2 BioMed Research International

calcitriol modifies neuromuscular and immune functions[13] Low Vit D3 levels are associated with skeletal fragilityand fractures Vit D3metabolites are major contributors ofbone andmineral homeostasis including the effect of 24R25-(OH)2D3on the osteoblastic differentiation of human MSCs[14] Bone homeostasis and repair are regulated by a networkof Vit D3metabolites Calcitriol the most biologically activeform of Vit D3 serves various physiological functions inthe body the most crucial being the maintenance of thecalcium and phosphorus balance which affects bone healthIn addition Vit D3 is essential for calcification [15] In a studyon 2-day-old Kunming white mice Vit C-p and Vit D3 sup-plements effectively promoted osteoblast differentiation inmouse embryoid bodies [16] Moreover calcitriol stimulatesthe in vitro differentiation of human MSCs into osteoblastswhich can be monitored on the basis of the increase in thealkaline phosphatase (ALP) enzyme activity or osteocalcin(OCN) gene expression [14] Few studies have investigatedthe dosendashresponse and dosendashtime effects of calcitriol onhuman P-MSCs for bone tissue engineering

The present study examined the effects of calcitriol-induced osteogenic differentiation on human alveolar perios-teum-derived MSCs (P-MSCs) and assessed the optimalcalcitriol concentration and incubation period required forosteogenic induction

2 Materials and Methods

21 Tissue Preparation Periosteal tissues were harvestedfrom 12 male and 18 female patients under 65 years witha mean age of 481 plusmn 123 during surgery at the dentaldepartment of Chang GungMemorial Hospital between 2011and 2013TheResearch andEthicsCommittee ofChangGungMemorial Hospital approved the study protocol All patientsprovided written informed consent

The harvested periosteal tissues were processed andstored inDulbeccorsquos phosphate-buffered saline (DPBSGibcoCarlsbad CA USA) with 300UmL of penicillin and300 120583gmL of streptomycin (Gibco BRL) and transferred toa laboratory within 4 hours where the tissues were manuallyminced using scalpels The obtained fragments were placedon 35-mm culture plates (Corning) containing 15mL ofgrowth medium 1 (120572-modified Eaglersquos medium [HyClone]10 fetal bovine serum [FBS Invitrogen] 300UmL ofpenicillin and 300 120583gmL of streptomycin) When theseperiosteum-derived cells (PDCs) reached subconfluence(80) the adherent cells were detached and separatedAnother growth medium (120572-modified Eaglersquos medium 10FBS 100UmL of penicillin and 100 120583gmL of streptomycin)was applied for cell culture The first replanted cells werelabeled P-MSC passage 1 Subsequent passages of cultured P-MSCs were achieved by following the same treatment proto-col when the previous passages reached 80 confluence

All assays were performed using P-MSC passages 3 to5 The induced osteogenic chondrogenic and adipogenicdifferentiation were evaluated through culture staining

22 Flow Cytometry In this experiment 1 times 106 cells wereharvested through 025 trypsin digestion and collected

through centrifugation Cell pellets were washed three timesusing 1x DPBS following which 1x permeabilization buffer(eBioscience San Diego CA) was added This buffer wasused to fix and permeabilize cells prior to the intracellularstaining of cytokines and other cytoplasmic and nuclearantigens Thereafter surface marker antibodies against CD19(FITC BD) CD34 (FITC BD) CD44 (PE BD) CD45(FITC BD) CD73 (PE BD) CD90 (APC BD) STRO-1(Alex BioLegend) and HLA-DR (FITC BD) were added tothe cells and incubated for 30 minutes at 4∘C in the darkThe cells were washed twice using 1x DPBS fixed using2 formaldehyde and analyzed using a FACSVerse flowcytometer (BD Biosciences) that measured 10000ndash20000cells per sample

23 Cell Attachment and Viability during Osteogenic Adi-pogenic and Chondrogenic Differentiation P-MSC sampleswere independently induced for differentiation into osteo-genic adipogenic and chondrogenic lineages Cells frompassages 3ndash5 were cultured on six-well plates with specificdifferentiation media containing differentiation-inducingreagents

For osteogenic differentiation the cells were cultured in120572-minimum essential medium (MEM) at a density of 5000cells per well on six-well plates On achieving approximately80 confluence the cells were cultured in an osteogenicmedium containing 120572-MEM (HyClone) 5 FBS (Invitro-gen) 120573-glycerophosphate (10mM Sigma) dexamethasone(100 nM Sigma) and 2-phospho-L-ascorbic acid trisodiumsalt (Vit C-p 100 120583M Sigma)Themedia were changed twiceper week To assess their potential to differentiate into theosteogenic lineage the cells were stained using von Kossawhich distinguishes calcified deposits present in a culture

For chondrogenic differentiation the cells were culturedat a density of 5000 cells per well on six-well plates in adifferentiation medium containing 120572-MEM (HyClone) 5FBS (Invitrogen) dexamethasone (100 nM Sigma) sodiumpyruvate (100 120583gmL Gibco) insulinndashtransferrinndashselenium-A (1x ITS 1-1884 Sigma) transforming growth factor-120573(10 ngmL RampD) and 5mL of 2-phospho-L-ascorbic acidtrisodium salt (Vit C-p 100 120583M Sigma) Furthermore thedifferentiation was evaluated according to the stained gly-cosaminoglycan in the culture identified through Alcian bluestaining For every cell sample subjected to differentiation acontrol culture of PDCs was maintained in 120572-MEM and 5FBS for 4-5 weeks and subjected to the same staining tests asthose for the experimental cells

For adipogenic differentiation the cells were cultured in120572-MEM at a density of 5000 cells per well on six-well platesOn achieving confluence the cells were cultured in a mediacontaining 120572-MEM (HyClone) 5 FBS (Invitrogen) 05mM3-isobutyl-1-methylxanthine (Sigma) dexamethasone (1 120583MSigma) insulin (5 120583gmL Sigma) and indomethacin (60120583MSigma)The presence of lipid droplets was evaluated usingOilRedO staining to determine whether cells were differentiatedinto the adipogenic lineage

24 Effects of Calcitriol on Osteogenesis Passages 3ndash5 ofP-MSCs were categorized into six groups according to

BioMed Research International 3

the culture media the control group [120572-MEM (HyClone)5 FBS (Invitrogen) 10mM of 120573-glycerophosphate (Sigma)and 10minus7Mdexamethasone (Sigma)] Vit C-p group (controlwith 100 120583M Vit C-p) 10minus10M calcitriol (Nang Kuang Phar-maceutical Co Ltd) group (control with 10minus10M calcitriol)10minus9M calcitriol group (control with 10minus9M calcitriol)10minus8M calcitriol group (control with 10minus8M calcitriol) and10minus7M calcitriol group (control with 10minus7M calcitriol)

25 Reverse Transcription and Quantitative Real-Time Poly-merase Chain Reaction After 7 days of osteoblast differ-entiation the Trizol reagent was used for isolating totalRNA 10 120583g of which was reverse-transcribed using avianmyeloblastosis virus reverse transcriptase (Roche)

First-strand complementary DNA (cDNA) was synthe-sized and quantitative polymerase chain reaction (qPCR)was performed using 5 ng120583L of cDNA Quantitative real-time (qRT)-PCR was conducted using primers for ALP BSPCBFA1 Col-1 OCN andVDR To avoidDNA contaminationby signals forward and reverse sequences of each primerwere designed on distinct exons qPCR was performed usingthe SYBR Green PCR Master Mix and TaqMan Master Mix(Applied Biosystems) according to manufacturer instruc-tions Furthermore the reactions were performed using theViiA7 Real-Time PCR system (Applied Biosystems) with theTaqMan Master Mix at 50∘C for 2min followed by 95∘C for10min and then 40 cycles each at 95∘C for 15 s and 60∘C for60 s The SYBR use was followed by PCR at 95∘C for 10minand then 40 cycles each at 95∘C for 15 s 60∘C for 60 s and60∘C for 15min The Ct values for ALP BSP CBFA1 Col-1VDR andOCNmessenger RNAs (mRNAs) were normalizedto the value of the housekeeping gene GAPDHmRNA

26 Effects of Vit C-p andVaryingCalcitriol Concentrations onOsteogenesis To confirm the differentiation of human PDCsinto a P-MSC-related osteoblast phenotype at the molecularlevel we monitored the expression of human ALP BSPCBFA1 Col-1 OCN andVDRmRNAs at the first and secondweeks through qRT-PCR by using human-specific primersThe results are presented as the fold change relative to thecontrol group results which was set to a value of 1

27 Primer Pairs Used for qRT-PCR Analysis The primer pairsequences used are mentioned in Table 1

28 Alkaline Phosphatase Activity Osteogenesis was quan-titated by measuring ALP activity Passages 3ndash5 P-MSCswere grown in the osteogenic medium the cultured cellswere using 100mM Tris and 1 Triton-100 Furthermorethe Alkaline Phosphatase Activity Colorimetric Assay Kit(BioVision) was used for determining the ALP activity atthe second and third week of culture by using a previouslydescribed method [17] This method employs the conversionof p-nitrophenyl phosphate to p-nitrophenol determined at awavelength of 450 nm and theALP activity is calculated froma standard value In this study the total p-nitrophenol formedwas normalized to total protein in 1 hour to determine theALP activity (ALP activityminmg protein)The fold change

Table 1 List of primers used in this study

Gene Primer designALP Hs01029144 m1

BSP Forward primer 51015840-AAAGTGAGAACGGGGAACCT-31015840

Reverse primer 51015840-GATGCAAAGCCAGAATGGAT-31015840

CBFA1 Hs00231692 m1

Col-1 Forward primer 51015840-CCTCAAGGGCTCCAACGAG-31015840

Reverse primer 51015840-TCAATCACTGTCTTGCCCCA-31015840

OCN Forward primer 51015840-GTGCAGCCTTTGTGTCCAAG-31015840

Reverse primer 51015840-GTCAGCCAACTCGTCACAGT-31015840

VDR Hs01045846 m1GAPDH Hs99999905 m1

activity in the experimental and control groups at the secondand third week of culture was also compared

Because the ALP mRNA test revealed that P-MSCssignificantly induced osteoblast differentiation at calcitriolconcentrations of 10minus8M and 10minus7M the ALP activity assaywas attempted using these concentrations The ALP activityof the control Vit C-p 10minus8Mcalcitriol and 10minus7Mcalcitriolgroups was assessed at the second and third weeks of culture

29 Statistical Analysis All values are represented as meansplusmn standard deviation (SD) Statistical significance was deter-mined using an unpaired Studentrsquos 119905-test and 119875 lt 005 wasconsidered statistically significant

3 Results

31 Cell Isolation Morphology and Osteogenic Chondrogenicand Adipogenic Differentiation Eventually we examined P-MSCs from two male and three female patients Under anoptical microscope the initial culture had small and roundcells before attachment and spindle-shaped cells thereafterPDCs proliferated in spherical colonies on days 1ndash14 Mor-phologically phase-contrast microscopy revealed that mostcells were spindle-shaped and that they formed a homoge-nous cell population (Figure 1) After the first passage thecells no longer proliferated in clusters but exhibited a uni-formly widespread pattern PDCs were spindle-shaped withirregular processes and firmly attached themselves to theculture plate at day 3 of the primary culture (Figure 1(a))Thereafter the cells exhibited a homogeneous fibroblast-likespindle shape (Figure 1)

At the end of the osteogenic differentiation von Kossastaining revealed the presence of calcium deposits andosteogenic differentiation for 4ndash6 weeks (Figure 1(b)) Pro-teoglycan production an indicator of chondrogenic dif-ferentiation was observed by staining PDCs with Alcianblue (Figure 1(c)) for 4ndash6 weeks For inducing adipogenicdifferentiation PDCs were cultured in a specific mediumas previously described [7] After 8 weeks of inductionPDC-derived adipocytes were stained using Oil Red O toconfirm the presence of intracellular lipids (Figure 1(d))


(a) (b)

(c) (d)

Figure 1 Differentiation study (a) Mesenchymal stem cells (MSCs) were spindle-shaped with irregular processes and firmly attached to theculture plate after 1ndash3 days of primary culture (asterisk) Under in vitro culture conditions MSCs were subpassaged and differentiated into(b) osteoblasts (asterisk black von Kossa staining) (c) chondrocytes (asterisk blue Alcian blue staining) and (d) adipocytes (asterisk pinkOil Red O staining)

The results revealed that PDCs could be decent subpassagedand potentially differentiated into P-MSCs

32 Flow Cytometric Surface Marker Expression Analysis forPDCs A flow cytometric assay was conducted for defin-ing the surface marker expression of MSCs before seedingthem into biomaterial scaffolds The PDCs tested positivefor the MSC markers CD73 (9818) CD90 (9994)STRO-1 (9983) and CD44 (9993) but negative for thehematopoietic lineage markers CD19 (109) CD34 (052)CD45 (135) and HLA-DR (047) (Figure 2)

33 Alkaline Phosphatase mRNA Expression at the First andSecond Weeks ALP mRNA a known marker for detectingearly osteogenic cell differentiation acts as an ectoenzymein the degradation of inorganic pyrophosphate for releasingphosphate for mineralization [18] Compared with the nega-tive control group the fold changes of ALPmRNA expressionof the first- and second-week cultures were 368 plusmn 224 and512 plusmn 334 for the Vit C-p group 093 plusmn 014 and 095 plusmn 021for the 10minus10M calcitriol group 159 plusmn 041 and 193 plusmn 061for the 10minus9M calcitriol group 450 plusmn 169 and 482 plusmn 244 forthe 10minus8M calcitriol group and 149 plusmn 037 and 488 plusmn 161for the 10minus7M calcitriol group respectively (Figure 1(a)) The10minus9 10minus8 and 10minus7M calcitriol and Vit C-p groups revealedsignificantly different ALP mRNA expression at both weeks(119875 lt 005 Figure 1(a)) Data pooled and analyzed at 2 weeksrevealed a similar developing pattern

The 10minus9 10minus8 and 10minus7M calcitriol groups revealedsignificant changes in ALP mRNA expression fold changescompared with the 10minus10M calcitriol group at the end ofboth weeks (119875 lt 005) The Vit C-p and 10minus7and 10minus8Mcalcitriol groups revealed a similar ALP mRNA expression atboth weeks however the 10minus7M and 10minus8M calcitriol groupsrevealed a significant variation at the first week (Figure 3(a)and Supplemental Table 1 available in Supplementary Mate-rial at httpdxdoiorg10115520163529561)

34 Bone Sialoprotein mRNA Expression at the First andSecond Week The fold changes of BSP mRNA expressionbetween the first and second weeks generally revealed a non-significant change for all tested groups (Table 2) Howeverthe fold changes of BSP mRNA expression mainly occurredat the first week they were 234plusmn059 152plusmn046 191plusmn074248 plusmn 095 and 225 plusmn 100 for the Vit C-p and 10minus10M10minus9M 10minus8M and 10minus7M calcitriol groups (SupplementalTable 2) At the second week only the 10minus7M calcitriol groupexhibited significantly higher BSPmRNAexpression than didthe control group (119875 lt 005 Figure 3(b)) Nonsignificantdifferences were observed between the Vit C-p and calcitriolgroups (Supplemental Table 2)

35 Core-Binding Factor Alpha-1 mRNA Expression atthe First and Second Weeks The fold changes of CBFA1mRNA expression between the first and second weeks werenonsignificant for most tested groups except for the 10minus7Mcalcitriol subgroups (119875 = 0009 Table 2) Vit C-p and 10minus8M


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Figure 2 Surface marker expression of mesenchymal stem cells through flow cytometry The surface markers expressed by mesenchymalstem cells mainly included CD73 (9818) CD90 (9994) STRO-1 (9983) and CD44 (9993) but not CD45 (135) CD34 (052)CD19 (109) or HLA-DR (047)

calcitriol significantly upregulated the CBFA1 mRNA expres-sion in cells at both weeks compared with the expression incontrols by contrast 10minus7Mcalcitriol upregulated the CBFA1mRNA expression only at the second week Differencesamong the three subgroups were nonsignificant (Figure 3(c)and Supplemental Table 3)

36 Collagen-1 mRNA Expression at the First and SecondWeeks The 10minus10 and 10minus9M calcitriol groups exhibitedslightly significant differences between the first and second

weeks (Table 2) Compared with the control groups VitC-p and 10minus9 10minus8 and 10minus7M calcitriol at the first weekand 10minus10 10minus9 10minus8 and 10minus7M calcitriol at the secondweek significantly increased the Col-1 mRNA expression(Figure 3(d) and Supplemental Table 4)

37 Osteocalcin mRNA Expression at the First and SecondWeeks OCN mRNA expressions did not significantly differbetween the first and second weeks for the Vit C-p and 10minus1010minus8 and 10minus7M calcitriol groups (Table 2) Vit C-p did not


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(f)

Figure 3 mRNA expressions of osteoblast differentiation for first and second weeks (a) ALP expression (b) BSP (c) CBFA1 (d) COL-I (e)OCN and (f) VDR


Table 2 Fold change significance of tested mRNAs (first week versus second week)

Group mRNAALP BSP CBFA1 Col-1 OCN VDR

Vit C 0445 0091 0898 0980 0925 043210minus10MD 0857 0624 0505 0044lowast 0303 090010minus9MD 0330 0102 0421 0038lowast 0043lowast 023510minus8MD 0813 0341 0535 0143 0473 032110minus7MD 0017lowast 0529 0009lowastlowast 0067 0871 0517lowast119901 lt 005 and lowastlowast119901 lt 001Exploring the statistical significance of the tested mRNAsrsquo fold change between the first and second weeks and determining if the mRNAs could be discussedby combining the two sets of data

Table 3 Fold change of ALP activity assays corresponding to calcitriol dose studies at the second and third weeks

Time points Fold change mean plusmn SD Vit C 119864 minus 8MD 119864 minus 7MD

Control 2W 002lowast 003lowast 0113W 004lowast 002lowast 002lowast

Vit C 2W 24 plusmn 110 088 1003W 301 plusmn 186 062 092

119864 minus 8MD 2W 228 plusmn 110 0913W 246 plusmn 109 053

119864 minus 7MD 2W 239 plusmn 175

3W 313 plusmn 166lowast119901 lt 005119864 minus 8MD 10minus8M calcitriol 119864 minus 7MD 10minus7M calcitriol Vit C ascorbic acid (100 120583M)

affect OCN mRNA expression in either week However the10minus8and 10minus7M calcitriol groups displayed a twofold increasein OCN mRNA expression at both weeks compared withthat of the control and Vit C-p groups (Figure 3(e) andSupplemental Table 5)

38 Vitamin D Receptor mRNA Expression at the First andSecond Weeks All tested groups presented nonsignificantVDRmRNA expression changes between the first and secondweeks (119875 gt 005 Table 2) Only the 10minus7M calcitriol grouprevealed a significant VDRmRNA expression which was 24times higher than that of other groups including the Vit C-pgroup at both weeks (Figure 3(f) and Supplemental Table 6)

39 ALP Enzyme Activity Assay mRNA examinationrevealed that the decisive calcitriol fold changes were associ-ated with 10minus8M and 10minus7M calcitriol therefore 10minus8Mand 10minus7M calcitriol were used to examine the ALP activity(activityminmg protein) at the second and third weeksThe fold change of osteogenesis-related ALP activity wasassessed in the control Vit C-p 10minus8M calcitriol and 10minus7Mcalcitriol groups (Table 3)

Time studies revealed nonsignificant variation in the foldchange of ALP activity between the second and third weeks(Figure 4) Compared with that in the control group foldchange in the Vit C-p 10minus8M calcitriol and 10minus7M calcitriolgroups increased by 240 plusmn 110 (119875 = 0021) 228 plusmn 110(119875 = 00033) and 239 plusmn 175 (119875 = 0114) times respectivelyin the second week and 301 plusmn 186 (119875 = 0042) 246 plusmn 109(119875 = 0018) and 313 plusmn 166 (119875 = 0023) times respectively

in the third weekThese results revealed that 10minus8Mcalcitrioland Vit C-p significantly enhanced the fold change of ALPactivity at the second and third weeks

4 Discussion

Tissue engineering by using MSCs is a recent therapeuticmodalityMSCs multipotent cells differentiating into variousfunctionalmesodermal tissue cell types [19] compose severaltissues types and can differentiate into various functionalcells including osteoblasts The periosteum serves as a nichefor progenitor cells and a rich vasculature supply for the boneit envelops [20] The proliferation rate of the periosteal cellswas higher than that of marrow stromal cells [21] and P-MSCswere ideal candidates for bone tissue regeneration [22]Furthermore P-MSCs are considered useful for reducing thecell culture time thus reducing both cost and contaminationrisk [21]

In this study the osteogenic chondrogenic and adi-pogenic differentiation of PDCs was induced under specificdifferentiation media This observation was in concordancewith that described in previous studies evaluating cellsextracted from the gingival tissue and bone marrow [23]The present study results comply with the minimal standardsrequired for the phenotypic and functional definition ofhuman MSCs as stated by the International Society forCellular Therapy [1] The results also reveal that P-MSCs canbe isolated and expanded revealing characteristics similar tothose typically described for bone marrow stromal cells


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Figure 4 Alkaline phosphatase activity assay of osteoblast differentiation for second and third weeks

Bone MSCs exhibited osteogenic differentiation withdexamethasone ascorbate and 120573-glycerophosphate [24]Calcitriol in combination with ascorbic acid promotes thedifferentiation of human MSCs into osteoblasts [25] Fur-thermore calcitriol is crucial for calcium homeostasis andbonemetabolism it influences the cardiovascular system butthe underlying mechanisms remain unclear [26] Thereforedexamethasone ascorbate and 120573-glycerophosphate wereincluded in this study for enhancing P-MSC differentiationMoreover according to our review of relevant literature thisis the first study revealing the osteogenic potential of P-MSCsusing different calcitriol concentrations by comparing theosteogenic gene expression

Human MSC trials using early passage cellular culturesmay avoid the senescent effects of expansion and reducethe cell culture period thus reducing both cost and con-tamination risk In accordance with previous osteogenicdifferentiation study [26] this study examined P-MSCs atpassages 3ndash5 and subsequent cultures for 2 weeks undercontrolled conditions

Calcitriol induced the expression of both early [6] andlate-stage markers of osteoblast differentiation in MSCsincluding ALP osteopontin BSP OCN mRNAs [24] andmatrix Gla protein in osteoblast-like cell cultures [27] Theeffects of varying calcitriol concentrations on osteogenesishave not been extensively discussed Therefore ascorbic acidand 10minus7 10minus8 10minus9 and 10minus10M calcitriol concentrationsregulated the ALP activity and osteogenic gene expression ofALP BSP CBFA1 Col-1 OCN and VDR mRNAs

Vit C-p considerably regulated the ALP and CBFA1mRNA expression and ALP activity at the first and secondweeks as well as BSP and Col-1 mRNA expression at the firstweek The 10minus10M calcitriol group revealed some changesin BSP and Col-1 mRNA expression at the first and secondweeks respectively (119875 lt 005 Supplemental Tables 2 and4) The 10minus9M calcitriol group mainly influenced ALP Col-1and OCN mRNA expression at both weeks and BSP mRNAexpression at the first week (119875 lt 005 Supplemental Tables1ndash6) Furthermore 10minus8M calcitriol majorly stimulated ALPCBFA1 Col-1 and OCN mRNA expression at both weeksand BSP mRNA expression at the first week (119875 lt 005 Sup-plemental Tables 1ndash6) A concentration of 10minus7M calcitriolintensified the fold change ofALP BSPOCNCol-1 andVDR

mRNA expression at both weeks as well as the CBFA1 mRNAexpression at the secondweek (119875 lt 005 Supplemental Tables1ndash6)

ALP BSP and CBFA1 mRNA expression was negativelycorrelated between the 10minus7 10minus8 and 10minus9Mcalcitriol groupsand the Vit C-p group at both weeks However the 10minus7Mcalcitriol group revealed a higher expression than did the VitC-p group with respect to OCN and VDRmRNA expressionat both weeks and Col-1 mRNA expression at the secondweek By contrast the OCNmRNA expression at both weekswas higher in the 10minus8M calcitriol group than in the Vit C-p group More trials are required to elucidate the effect ofcalcitriol on osteogenic induction

Dose studies revealed that 10minus7Mcalcitriol and not otherexamined calcitriol concentrations highly enhanced VDRmRNA expression at both tested weeks moreover comparedwith 10minus8Mcalcitriol 10minus7Mcalcitriol weakly expressed ALPmRNAAfter time studies revealed nonsignificant differencesbetween the first and second weeks we applied the averagevalues of both weeks for analyzing the developing patterns(Supplemental Tables 1ndash6) Therefore the 10minus7and 10minus8Mcalcitriol groups exhibited higher ALP CBFA1 and OCNmRNA expression than did the 10minus9M calcitriol group atboth weeks a similar effect was observed for Col-1 mRNAexpression at the second week

ALP OCN and Col-1 are markers capable of detectingearly osteogenic cell differentiation [28] by contrast ALP actsas an ectoenzyme in the degradation of inorganic pyrophos-phate for releasing phosphate for mineralization [18] OCNa major noncollagen bone protein is a crucial osteogenicmarker regulating the formation of mineral nodules causingosteogenesis [29] Col-1 is the most abundant matrix proteinand OCN regulates matrix synthesis [30] Both OCN andCol-1 are acidic bone matrix proteins with crucial roles inmatrix synthesis and are characteristic of mature matrix-forming osteoblasts However OCN osteopontin and BSPexpression are considered the mature osteogenic markers[28 31 32] Calcitriol significantly upregulatedOCNandCol-1 mRNA expression [14 25 33] and did not substantiallyaffect CBFA1 or ALP gene expression [14] However ourresults revealed that 10minus7and 10minus8M calcitriol upregulatedosteogenesis by increasing ALP BSP CBFA1 Col-1 andOCN mRNA expression at both weeks Moreover VDR


a hormone-regulated transcription factor that interacts withcoactivators and corepressors is associated with chromatinhistone modifications and transcriptional regulation [34]Similar to a previous study stating that calcitriol increasesVDRmRNA [14] We confirmed that 10minus7M calcitriol signif-icantly stimulates VDR mRNA expression

According to the National Institutes of Health 20ndash50 ngmL (50ndash125 nmolL) of serum 25-hydroxyvitamin D is desir-able but concentrations exceeding 30 ngmL (75 nmolL)are not consistently associated with an increased benefitSerum 25-hydroxyvitamin D levels exceeding 50 ngmL(125 nmolL) may cause complications However this studyindicated that 10acircĂŠ7M which equals 100 nmolL calcitriolsignificantly upregulates VDRmRNA Additional studies arerequired to explore the advantages and disadvantages of highcalcitriol concentrations

In contrast to a previous study which reported that ALPactivity was inhibited by tumor-growing factor-beta (01ndash10 ngmL) and increased by calcitriol (50 nM which equals5 times 10minus8M) [33] our results revealed that 10minus7 and 10minus8Mcalcitriol and Vit C-p enhanced P-MSC-related ALP activity

The current results clearly demonstrate the feasibilityof isolating MSCs from the periosteum and differentiatingthem into osteoblasts chondrocytes and adipocytes Alveo-lar periosteum is rich in MSCs which can be easily obtainedfrom routine oral surgery and poses fewer complicationsthus they are suitable candidate donor sites for tissue engi-neering

This time- and dose-based study assumed that Vit C-p and 10minus7and 10minus8M calcitriol share some mechanisms forinducing osteogenesis by increasing the ALP activity andALP BSP CBFA1 and Col-1 mRNA expression Moreover10minus8 and 10minus7M calcitriol have more potential osteoinductiv-ity than does 10minus9M calcitriol on alveolar P-MSCs Calcitriolexclusively induces OCN and VDR mRNA osteoinduction

Consistent with the results of some previousMSC studiesVit C-p and calcitriol significantly enhanced BSP mRNA atthe first week however only 10minus7M calcitriol significantlyupregulated BSP mRNA at the second week in this studyThe small sample size and the nature of the P-MSCs possiblyplayed a role on this variation In addition the nonsignificantdifference between the first 2 weeks can be partially explainedby the fact that BSP is considered a mature osteoblast markerIn the future we plan to increase the tested sample numberand evaluate the P-MSC cultures for changes in BSP mRNAat later stages of in vitro differentiation and maturation

5 Conclusion

In summary alveolar periosteum is rich in MSCs HumanP-MSCs can be induced to proliferate and differentiate intoan osteogenic lineage There is a trend showing that P-MSCstreated with Vit C-p and 10minus9 10minus8 and 10minus7M calcitriolpromote ALP activity as well as the osteogenic differen-tiation of ALP BSP CBFA1 and Col-1 mRNA expressionBy contrast 10minus8and 10minus7M calcitriol enhanced osteogenicOCN mRNA expression significantly more than did Vit C-p The 10minus7M calcitriol group exhibited upregulated VDR

mRNA expression Further investigation is required into thesignificance of the osteoinductivemechanismbetweenVit C-p and calcitriol

Competing Interests

The authors declare that there is no conflict of interests

Authorsrsquo Contributions

Hsiang-Hsi Hong was responsible of conception and designstudymaterials data collection andmanuscript writing Yen-LiWangwas responsible of administrative support data anal-ysis and interpretation and final approval of the manuscriptAdrienne Hong and Tzung-Hai Yen were responsible of dataassembly and analysis and manuscript writing

Acknowledgments

The study protocol was approved by the Institutional ReviewBoard for Clinical Research of Chang Gung MemorialHospital (IRB99-1828B 100-3019C 99-3814B 102-1619C 101-4728B and 103-4223C) This study was supported by ChangGung Memorial Hospital (CMRPG392071 CMRPG3A1141CMRPG3A1142 and NMRPG3C0151)

References

[1] M Dominici K Le Blanc I Mueller et al ldquoMinimal crite-ria for defining multipotent mesenchymal stromal cells Theinternational society for cellular therapy position statementrdquoCytotherapy vol 8 no 4 pp 315ndash317 2006

[2] S Gronthos M Mankani J Brahim P G Robey and S ShildquoPostnatal human dental pulp stem cells (DPSCs) in vitro andin vivordquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 25 pp 13625ndash13630 2000

[3] M Miura S Gronthos M Zhao et al ldquoSHED Stem cells fromhuman exfoliated deciduous teethrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 100 no10 pp 5807ndash5812 2003

[4] B M Seo M Miura S Gronthos et al ldquoInvestigation of multi-potent postnatal stem cells from human periodontal ligamentrdquoThe Lancet vol 364 no 9429 pp 149ndash155 2004

[5] W Sonoyama Y Liu D Fang et al ldquoMesenchymal stem cell-mediated functional tooth regeneration in swinerdquo PLoS ONEvol 1 no 1 article e79 2006

[6] C Morsczeck W Gotz J Schierholz et al ldquoIsolation ofprecursor cells (PCs) from human dental follicle of wisdomteethrdquoMatrix Biology vol 24 no 2 pp 155ndash165 2005

[7] T I Mitrano M S Grob F Carrion et al ldquoCulture and char-acterization of mesenchymal stem cells from human gingivaltissuerdquo Journal of Periodontology vol 81 no 6 pp 917ndash9252010

[8] C A Squier S Ghoneim and C R Kremenak ldquoUltrastructureof the periosteum from membrane bonerdquo Journal of Anatomyvol 171 pp 233ndash239 1990

[9] J Aubin and J Triffitt ldquoMesenchymal stem cells and osteoblastdifferentiationrdquo in Principles of Bone Biology J Bilezikian L GRaisz and G A Rodan Eds pp 59ndash81 Academic Press SanDiego Calif USA 2002


[10] L Diaz-Flores R Gutierrez A Lopez-Alonso R Gonzalez andH Varela ldquoPericytes as a supplementary source of osteoblastsin periosteal osteogenesisrdquo Clinical Orthopaedics and RelatedResearch no 275 pp 280ndash286 1992

[11] Y-S Choi S-E Noh S-M Lim et al ldquoMultipotency andgrowth characteristic of periosteum-derived progenitor cellsfor chondrogenic osteogenic and adipogenic differentiationrdquoBiotechnology Letters vol 30 no 4 pp 593ndash601 2008

[12] S H McBride S Dolejs S Brianza U Knothe and M LKnothe Tate ldquoNet change in periosteal strain during stanceshift loading after surgery correlates to rapid de novo bonegeneration in critically sized defectsrdquo Annals of BiomedicalEngineering vol 39 no 5 pp 1570ndash1581 2011

[13] Office of Dietary Supplements (ODS) National Institutesof Health (NIH) httpsodsodnihgovfactsheetsVitaminD-HealthProfessional

[14] K M Curtis K K Aenlle B A Roos and G A Howardldquo24R25-Dihydroxyvitamin D3 promotes the osteoblastic dif-ferentiation of human mesenchymal stem cellsrdquo MolecularEndocrinology vol 28 no 5 pp 644ndash658 2014

[15] N Z Mostafa R Fitzsimmons P W Major et al ldquoOsteogenicdifferentiation of humanmesenchymal stem cells cultured withdexamethasone vitamin D3 basic fibroblast growth factor andbone morphogenetic protein-2rdquo Connective Tissue Researchvol 53 no 2 pp 117ndash131 2012

[16] Y Sun X Yang F Li et al ldquoStudy on differentiation ofembryonic stem cells into osteoblast in vitro inducing by125(OH)2VD3rdquo Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhivol 22 no 9 pp 1117ndash1120 2008

[17] G DrsquoIppolito P C Schiller C Ricordi B A Roos and GA Howard ldquoAge-related osteogenic potential of mesenchymalstromal stem cells from human vertebral bonemarrowrdquo Journalof Bone and Mineral Research vol 14 no 7 pp 1115ndash1122 1999

[18] U Stucki J Schmid C F Hammerle andN P Lang ldquoTemporaland local appearance of alkaline phosphatase activity in earlystages of guided bone regeneration a descriptive histochemicalstudy in humansrdquo Clinical Oral Implants Research vol 12 no 2pp 121ndash127 2001

[19] A I Caplan and S P Bruder ldquoMesenchymal stem cells buildingblocks for molecular medicine in the 21st centuryrdquo Trends inMolecular Medicine vol 7 no 6 pp 259ndash264 2001

[20] M L Knothe Tate T D Falls S H McBride R Atit and U RKnothe ldquoMechanical modulation of osteochondroprogenitorcell faterdquo International Journal of Biochemistry and Cell Biologyvol 40 no 12 pp 2720ndash2738 2008

[21] H Agata I Asahina Y Yamazaki et al ldquoEffective boneengineering with periosteum-derived cellsrdquo Journal of DentalResearch vol 86 no 1 pp 79ndash83 2007

[22] O Hayashi Y Katsube M Hirose H Ohgushi and H ItoldquoComparison of osteogenic ability of rat mesenchymal stemcells from bone marrow periosteum and adipose tissuerdquoCalcified Tissue International vol 82 no 3 pp 238ndash247 2008

[23] M F Pittenger A M Mackay S C Beck et al ldquoMultilineagepotential of adult human mesenchymal stem cellsrdquo Science vol284 no 5411 pp 143ndash147 1999

[24] N R Joslashrgensen Z Henriksen O H Soslashrensen and RCivitelli ldquoDexamethasone BMP-2 and 125-dihydroxyvitaminD enhance a more differentiated osteoblast phenotype vali-dation of an in vitro model for human bone marrow-derivedprimary osteoblastsrdquo Steroids vol 69 no 4 pp 219ndash226 2004

[25] P Lui B O Oyajobi R G G Russell and A Scutt ldquoRegulationof osteogenic differentiation of human bone marrow stromal

cells interaction between transforming growth factor-120573 and125(OH)2 Vitamin D3 in vitrordquo Calcified Tissue Internationalvol 65 no 2 pp 173ndash180 1999

[26] J H Lee J H OrsquoKeefe D Bell D D Hensrud andM F HolickldquoVitamin D Deficiency An important common and easilytreatable cardiovascular risk factorrdquo Journal of the AmericanCollege of Cardiology vol 52 no 24 pp 1949ndash1956 2008

[27] P A Price and S A Baukol ldquo125-dihydroxyvitamin D3increases synthesis of the vitamin K-dependent bone protein byosteosarcoma cellsrdquo Journal of Biological Chemistry vol 255 no24 pp 11660ndash11663 1980

[28] D J Corn Y Kim M D Krebs et al ldquoImaging early stageosteogenic differentiation of mesenchymal stem cellsrdquo Journalof Orthopaedic Research vol 31 no 6 pp 871ndash879 2013

[29] X Shi Y Wang R R Varshney L Ren F Zhang andD-A Wang ldquoIn-vitro osteogenesis of synovium stem cellsinduced by controlled release of bisphosphate additives frommicrospherical mesoporous silica compositerdquo Biomaterials vol30 no 23-24 pp 3996ndash4005 2009

[30] R Fujisawa and M Tamura ldquoAcidic bone matrix proteins andtheir roles in calcificationrdquo Frontiers in Bioscience vol 17 no 5pp 1891ndash1903 2012

[31] P Alberius and M Gordh ldquoOsteopontin and bone sialopro-tein distribution at the bone graft recipient siterdquo Archives ofOtolaryngologymdashHead and Neck Surgery vol 124 no 12 pp1382ndash1386 1998

[32] S Dacic I Kalajzic D Visnjic A C Lichtler and D WRowe ldquoCol1a1-driven transgenic markers of osteoblast lineageprogressionrdquo Journal of Bone and Mineral Research vol 16 no7 pp 1228ndash1236 2001

[33] M van Driel M Koedam C J Buurman et al ldquoEvidencethat both 112057225-dihydroxyvitamin D3 and 24-hydroxylated D3enhance human osteoblast differentiation and mineralizationrdquoJournal of Cellular Biochemistry vol 99 no 3 pp 922ndash9352006

[34] Y Sakaguchi I Sekiya K Yagishita and T Muneta ldquoCompar-ison of human stem cells derived from various mesenchymaltissues superiority of synovium as a cell sourcerdquo Arthritis andRheumatism vol 52 no 8 pp 2521ndash2529 2005

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


calcitriol modifies neuromuscular and immune functions[13] Low Vit D3 levels are associated with skeletal fragilityand fractures Vit D3metabolites are major contributors ofbone andmineral homeostasis including the effect of 24R25-(OH)2D3on the osteoblastic differentiation of human MSCs[14] Bone homeostasis and repair are regulated by a networkof Vit D3metabolites Calcitriol the most biologically activeform of Vit D3 serves various physiological functions inthe body the most crucial being the maintenance of thecalcium and phosphorus balance which affects bone healthIn addition Vit D3 is essential for calcification [15] In a studyon 2-day-old Kunming white mice Vit C-p and Vit D3 sup-plements effectively promoted osteoblast differentiation inmouse embryoid bodies [16] Moreover calcitriol stimulatesthe in vitro differentiation of human MSCs into osteoblastswhich can be monitored on the basis of the increase in thealkaline phosphatase (ALP) enzyme activity or osteocalcin(OCN) gene expression [14] Few studies have investigatedthe dosendashresponse and dosendashtime effects of calcitriol onhuman P-MSCs for bone tissue engineering

The present study examined the effects of calcitriol-induced osteogenic differentiation on human alveolar perios-teum-derived MSCs (P-MSCs) and assessed the optimalcalcitriol concentration and incubation period required forosteogenic induction

2 Materials and Methods

21 Tissue Preparation Periosteal tissues were harvestedfrom 12 male and 18 female patients under 65 years witha mean age of 481 plusmn 123 during surgery at the dentaldepartment of Chang GungMemorial Hospital between 2011and 2013TheResearch andEthicsCommittee ofChangGungMemorial Hospital approved the study protocol All patientsprovided written informed consent

The harvested periosteal tissues were processed andstored inDulbeccorsquos phosphate-buffered saline (DPBSGibcoCarlsbad CA USA) with 300UmL of penicillin and300 120583gmL of streptomycin (Gibco BRL) and transferred toa laboratory within 4 hours where the tissues were manuallyminced using scalpels The obtained fragments were placedon 35-mm culture plates (Corning) containing 15mL ofgrowth medium 1 (120572-modified Eaglersquos medium [HyClone]10 fetal bovine serum [FBS Invitrogen] 300UmL ofpenicillin and 300 120583gmL of streptomycin) When theseperiosteum-derived cells (PDCs) reached subconfluence(80) the adherent cells were detached and separatedAnother growth medium (120572-modified Eaglersquos medium 10FBS 100UmL of penicillin and 100 120583gmL of streptomycin)was applied for cell culture The first replanted cells werelabeled P-MSC passage 1 Subsequent passages of cultured P-MSCs were achieved by following the same treatment proto-col when the previous passages reached 80 confluence

All assays were performed using P-MSC passages 3 to5 The induced osteogenic chondrogenic and adipogenicdifferentiation were evaluated through culture staining

22 Flow Cytometry In this experiment 1 times 106 cells wereharvested through 025 trypsin digestion and collected

through centrifugation Cell pellets were washed three timesusing 1x DPBS following which 1x permeabilization buffer(eBioscience San Diego CA) was added This buffer wasused to fix and permeabilize cells prior to the intracellularstaining of cytokines and other cytoplasmic and nuclearantigens Thereafter surface marker antibodies against CD19(FITC BD) CD34 (FITC BD) CD44 (PE BD) CD45(FITC BD) CD73 (PE BD) CD90 (APC BD) STRO-1(Alex BioLegend) and HLA-DR (FITC BD) were added tothe cells and incubated for 30 minutes at 4∘C in the darkThe cells were washed twice using 1x DPBS fixed using2 formaldehyde and analyzed using a FACSVerse flowcytometer (BD Biosciences) that measured 10000ndash20000cells per sample

23 Cell Attachment and Viability during Osteogenic Adi-pogenic and Chondrogenic Differentiation P-MSC sampleswere independently induced for differentiation into osteo-genic adipogenic and chondrogenic lineages Cells frompassages 3ndash5 were cultured on six-well plates with specificdifferentiation media containing differentiation-inducingreagents

For osteogenic differentiation the cells were cultured in120572-minimum essential medium (MEM) at a density of 5000cells per well on six-well plates On achieving approximately80 confluence the cells were cultured in an osteogenicmedium containing 120572-MEM (HyClone) 5 FBS (Invitro-gen) 120573-glycerophosphate (10mM Sigma) dexamethasone(100 nM Sigma) and 2-phospho-L-ascorbic acid trisodiumsalt (Vit C-p 100 120583M Sigma)Themedia were changed twiceper week To assess their potential to differentiate into theosteogenic lineage the cells were stained using von Kossawhich distinguishes calcified deposits present in a culture

For chondrogenic differentiation the cells were culturedat a density of 5000 cells per well on six-well plates in adifferentiation medium containing 120572-MEM (HyClone) 5FBS (Invitrogen) dexamethasone (100 nM Sigma) sodiumpyruvate (100 120583gmL Gibco) insulinndashtransferrinndashselenium-A (1x ITS 1-1884 Sigma) transforming growth factor-120573(10 ngmL RampD) and 5mL of 2-phospho-L-ascorbic acidtrisodium salt (Vit C-p 100 120583M Sigma) Furthermore thedifferentiation was evaluated according to the stained gly-cosaminoglycan in the culture identified through Alcian bluestaining For every cell sample subjected to differentiation acontrol culture of PDCs was maintained in 120572-MEM and 5FBS for 4-5 weeks and subjected to the same staining tests asthose for the experimental cells

For adipogenic differentiation the cells were cultured in120572-MEM at a density of 5000 cells per well on six-well platesOn achieving confluence the cells were cultured in a mediacontaining 120572-MEM (HyClone) 5 FBS (Invitrogen) 05mM3-isobutyl-1-methylxanthine (Sigma) dexamethasone (1 120583MSigma) insulin (5 120583gmL Sigma) and indomethacin (60120583MSigma)The presence of lipid droplets was evaluated usingOilRedO staining to determine whether cells were differentiatedinto the adipogenic lineage

24 Effects of Calcitriol on Osteogenesis Passages 3ndash5 ofP-MSCs were categorized into six groups according to












CBFA1 Hs00231692 m1





VDR Hs01045846 m1GAPDH Hs99999905 m1




3 Results




(a) (b)

(c) (d)









9818

CD73

0

20

40

60

80

100

o

f Max

9993

101 102 103100

STRO-1

0

20

40

60

80

100

o

f Max

135

101 102 103100

CD45

0

20

40

60

80

100

o

f Max

047

HLA-DR101 102 103100

0

20

40

60

80

100

o

f Max

109

101 102 103100

CD19

0

20

40

60

80

100

o

f Max

052

101 102 103100

CD34

0

20

40

60

80

100

o

f Max

9994

101 102 103100101 102 103100

CD90

0

20

40

60

80

100

o

f Max

9983

CD44

101 102 103100

0

20

40

60

80

100

o

f Max







100

368

093

159

450

149

1

512

095

193

482488

Con

trol

Vit

C

ALPGAPDH

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

012345678

mRN

A fo

ld ch

ange

1W2W

Eminus7

MD

Eminus8

MD

Eminus9

MD

Eminus10

MD

(a)

100

234

152

191

248 225

1

142134

121

179 188

BSPGAPDHlowast

lowastlowast

lowastlowast

lowast

0

05

1

15

2

25

3

35

mRN

A fo

ld ch

ange

1W2W

Con

trol

Vit

C

Eminus7

MD

Eminus8

MD

Eminus9

MD

Eminus10

MD

(b)

100

142

104 102

148

116

1

145

111 11

167 179

CBFA1GAPDHlowast

lowastlowast

lowastlowast

0

05

1

15

2

25

mRN

A fo

ld ch

ange

1W2W

Con

trol

Vit

C

Eminus7

MD

Eminus8

MD

Eminus9

MD

Eminus10

MD

(c)

1

1914944214

0975132809 1201022152

19539794531971530316

1

190079245

1355522893 1460983923

3073703112

4979549793

COL-IGAPDH

lowast

lowast

lowastlowast lowast

lowast

lowastlowast

0

1

2

3

4

5

6

7m

RNA

fold

chan

ge

1W2W

Con

trol

Vit

C

Eminus7

MD

Eminus8

MD

Eminus9

MD

Eminus10

MD

(d)

100 111 102 109

179

224

100 113

120 126

201

235

OCNGAPDH

lowast

lowast

lowast

lowastlowast

lowast

0

05

1

15

2

25

3

35

mRN

A fo

ld ch

ange

1W2W

Con

trol

Vit

C

Eminus7

MD

Eminus8

MD

Eminus9

MD

Eminus10

MD

(e)

100 101 097 088

116

272

100 093 096 101 100

237


0

05

1

15

2

25

3

35

mRN

A fo

ld ch

ange

1W2W

Con

trol

Vit

C

Eminus7

MD

Eminus8

MD

Eminus9

MD

Eminus10

MD

(f)


















4 Discussion




100

301

246

313

Control

lowastlowast

lowast

100

240 228

239 lowast

lowast



1

2

3

4

5

6

Fold

chan

ge

005

115

225

335

445

Fold

chan

ge

















5 Conclusion



Competing Interests




Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























CBFA1 Hs00231692 m1





VDR Hs01045846 m1GAPDH Hs99999905 m1




3 Results




(a) (b)

(c) (d)









9818

CD73

0

20

40

60

80

100

o

f Max

9993

101 102 103100

STRO-1

0

20

40

60

80

100

o

f Max

135

101 102 103100

CD45

0

20

40

60

80

100

o

f Max

047

HLA-DR101 102 103100

0

20

40

60

80

100

o

f Max

109

101 102 103100

CD19

0

20

40

60

80

100

o

f Max

052

101 102 103100

CD34

0

20

40

60

80

100

o

f Max

9994

101 102 103100101 102 103100

CD90

0

20

40

60

80

100

o

f Max

9983

CD44

101 102 103100

0

20

40

60

80

100

o

f Max







100

368

093

159

450

149

1

512

095

193

482488

Con

trol

Vit

C

ALPGAPDH

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

012345678

mRN

A fo

ld ch

ange

1W2W

Eminus7

MD

Eminus8

MD

Eminus9

MD

Eminus10

MD

(a)

100

234

152

191

248 225

1

142134

121

179 188

BSPGAPDHlowast

lowastlowast

lowastlowast

lowast

0

05

1

15

2

25

3

35

mRN

A fo

ld ch

ange

1W2W

Con

trol

Vit

C

Eminus7

MD

Eminus8

MD

Eminus9

MD

Eminus10

MD

(b)

100

142

104 102

148

116

1

145

111 11

167 179

CBFA1GAPDHlowast

lowastlowast

lowastlowast

0

05

1

15

2

25

mRN

A fo

ld ch

ange

1W2W

Con

trol

Vit

C

Eminus7

MD

Eminus8

MD

Eminus9

MD

Eminus10

MD

(c)

1

1914944214

0975132809 1201022152

19539794531971530316

1

190079245

1355522893 1460983923

3073703112

4979549793

COL-IGAPDH

lowast

lowast

lowastlowast lowast

lowast

lowastlowast

0

1

2

3

4

5

6

7m

RNA

fold

chan

ge

1W2W

Con

trol

Vit

C

Eminus7

MD

Eminus8

MD

Eminus9

MD

Eminus10

MD

(d)

100 111 102 109

179

224

100 113

120 126

201

235

OCNGAPDH

lowast

lowast

lowast

lowastlowast

lowast

0

05

1

15

2

25

3

35

mRN

A fo

ld ch

ange

1W2W

Con

trol

Vit

C

Eminus7

MD

Eminus8

MD

Eminus9

MD

Eminus10

MD

(e)

100 101 097 088

116

272

100 093 096 101 100

237


0

05

1

15

2

25

3

35

mRN

A fo

ld ch

ange

1W2W

Con

trol

Vit

C

Eminus7

MD

Eminus8

MD

Eminus9

MD

Eminus10

MD

(f)


















4 Discussion




100

301

246

313

Control

lowastlowast

lowast

100

240 228

239 lowast

lowast



1

2

3

4

5

6

Fold

chan

ge

005

115

225

335

445

Fold

chan

ge

















5 Conclusion



Competing Interests




Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)









9818

CD73

0

20

40

60

80

100

o

f Max

9993

101 102 103100

STRO-1

0

20

40

60

80

100

o

f Max

135

101 102 103100

CD45

0

20

40

60

80

100

o

f Max

047

HLA-DR101 102 103100

0

20

40

60

80

100

o

f Max

109

101 102 103100

CD19

0

20

40

60

80

100

o

f Max

052

101 102 103100

CD34

0

20

40

60

80

100

o

f Max

9994

101 102 103100101 102 103100

CD90

0

20

40

60

80

100

o

f Max

9983

CD44

101 102 103100

0

20

40

60

80

100

o

f Max







100

368

093

159

450

149

1

512

095

193

482488

Con

trol

Vit

C

ALPGAPDH

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

012345678

mRN

A fo

ld ch

ange

1W2W

Eminus7

MD

Eminus8

MD

Eminus9

MD

Eminus10

MD

(a)

100

234

152

191

248 225

1

142134

121

179 188

BSPGAPDHlowast

lowastlowast

lowastlowast

lowast

0

05

1

15

2

25

3

35

mRN

A fo

ld ch

ange

1W2W

Con

trol

Vit

C

Eminus7

MD

Eminus8

MD

Eminus9

MD

Eminus10

MD

(b)

100

142

104 102

148

116

1

145

111 11

167 179

CBFA1GAPDHlowast

lowastlowast

lowastlowast

0

05

1

15

2

25

mRN

A fo

ld ch

ange

1W2W

Con

trol

Vit

C

Eminus7

MD

Eminus8

MD

Eminus9

MD

Eminus10

MD

(c)

1

1914944214

0975132809 1201022152

19539794531971530316

1

190079245

1355522893 1460983923

3073703112

4979549793

COL-IGAPDH

lowast

lowast

lowastlowast lowast

lowast

lowastlowast

0

1

2

3

4

5

6

7m

RNA

fold

chan

ge

1W2W

Con

trol

Vit

C

Eminus7

MD

Eminus8

MD

Eminus9

MD

Eminus10

MD

(d)

100 111 102 109

179

224

100 113

120 126

201

235

OCNGAPDH

lowast

lowast

lowast

lowastlowast

lowast

0

05

1

15

2

25

3

35

mRN

A fo

ld ch

ange

1W2W

Con

trol

Vit

C

Eminus7

MD

Eminus8

MD

Eminus9

MD

Eminus10

MD

(e)

100 101 097 088

116

272

100 093 096 101 100

237


0

05

1

15

2

25

3

35

mRN

A fo

ld ch

ange

1W2W

Con

trol

Vit

C

Eminus7

MD

Eminus8

MD

Eminus9

MD

Eminus10

MD

(f)


















4 Discussion




100

301

246

313

Control

lowastlowast

lowast

100

240 228

239 lowast

lowast



1

2

3

4

5

6

Fold

chan

ge

005

115

225

335

445

Fold

chan

ge

















5 Conclusion



Competing Interests




Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















9818

CD73

0

20

40

60

80

100

o

f Max

9993

101 102 103100

STRO-1

0

20

40

60

80

100

o

f Max

135

101 102 103100

CD45

0

20

40

60

80

100

o

f Max

047

HLA-DR101 102 103100

0

20

40

60

80

100

o

f Max

109

101 102 103100

CD19

0

20

40

60

80

100

o

f Max

052

101 102 103100

CD34

0

20

40

60

80

100

o

f Max

9994

101 102 103100101 102 103100

CD90

0

20

40

60

80

100

o

f Max

9983

CD44

101 102 103100

0

20

40

60

80

100

o

f Max







100

368

093

159

450

149

1

512

095

193

482488

Con

trol

Vit

C

ALPGAPDH

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

012345678

mRN

A fo

ld ch

ange

1W2W

Eminus7

MD

Eminus8

MD

Eminus9

MD

Eminus10

MD

(a)

100

234

152

191

248 225

1

142134

121

179 188

BSPGAPDHlowast

lowastlowast

lowastlowast

lowast

0

05

1

15

2

25

3

35

mRN

A fo

ld ch

ange

1W2W

Con

trol

Vit

C

Eminus7

MD

Eminus8

MD

Eminus9

MD

Eminus10

MD

(b)

100

142

104 102

148

116

1

145

111 11

167 179

CBFA1GAPDHlowast

lowastlowast

lowastlowast

0

05

1

15

2

25

mRN

A fo

ld ch

ange

1W2W

Con

trol

Vit

C

Eminus7

MD

Eminus8

MD

Eminus9

MD

Eminus10

MD

(c)

1

1914944214

0975132809 1201022152

19539794531971530316

1

190079245

1355522893 1460983923

3073703112

4979549793

COL-IGAPDH

lowast

lowast

lowastlowast lowast

lowast

lowastlowast

0

1

2

3

4

5

6

7m

RNA

fold

chan

ge

1W2W

Con

trol

Vit

C

Eminus7

MD

Eminus8

MD

Eminus9

MD

Eminus10

MD

(d)

100 111 102 109

179

224

100 113

120 126

201

235

OCNGAPDH

lowast

lowast

lowast

lowastlowast

lowast

0

05

1

15

2

25

3

35

mRN

A fo

ld ch

ange

1W2W

Con

trol

Vit

C

Eminus7

MD

Eminus8

MD

Eminus9

MD

Eminus10

MD

(e)

100 101 097 088

116

272

100 093 096 101 100

237


0

05

1

15

2

25

3

35

mRN

A fo

ld ch

ange

1W2W

Con

trol

Vit

C

Eminus7

MD

Eminus8

MD

Eminus9

MD

Eminus10

MD

(f)


















4 Discussion




100

301

246

313

Control

lowastlowast

lowast

100

240 228

239 lowast

lowast



1

2

3

4

5

6

Fold

chan

ge

005

115

225

335

445

Fold

chan

ge

















5 Conclusion



Competing Interests




Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















100

368

093

159

450

149

1

512

095

193

482488

Con

trol

Vit

C

ALPGAPDH

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

012345678

mRN

A fo

ld ch

ange

1W2W

Eminus7

MD

Eminus8

MD

Eminus9

MD

Eminus10

MD

(a)

100

234

152

191

248 225

1

142134

121

179 188

BSPGAPDHlowast

lowastlowast

lowastlowast

lowast

0

05

1

15

2

25

3

35

mRN

A fo

ld ch

ange

1W2W

Con

trol

Vit

C

Eminus7

MD

Eminus8

MD

Eminus9

MD

Eminus10

MD

(b)

100

142

104 102

148

116

1

145

111 11

167 179

CBFA1GAPDHlowast

lowastlowast

lowastlowast

0

05

1

15

2

25

mRN

A fo

ld ch

ange

1W2W

Con

trol

Vit

C

Eminus7

MD

Eminus8

MD

Eminus9

MD

Eminus10

MD

(c)

1

1914944214

0975132809 1201022152

19539794531971530316

1

190079245

1355522893 1460983923

3073703112

4979549793

COL-IGAPDH

lowast

lowast

lowastlowast lowast

lowast

lowastlowast

0

1

2

3

4

5

6

7m

RNA

fold

chan

ge

1W2W

Con

trol

Vit

C

Eminus7

MD

Eminus8

MD

Eminus9

MD

Eminus10

MD

(d)

100 111 102 109

179

224

100 113

120 126

201

235

OCNGAPDH

lowast

lowast

lowast

lowastlowast

lowast

0

05

1

15

2

25

3

35

mRN

A fo

ld ch

ange

1W2W

Con

trol

Vit

C

Eminus7

MD

Eminus8

MD

Eminus9

MD

Eminus10

MD

(e)

100 101 097 088

116

272

100 093 096 101 100

237


0

05

1

15

2

25

3

35

mRN

A fo

ld ch

ange

1W2W

Con

trol

Vit

C

Eminus7

MD

Eminus8

MD

Eminus9

MD

Eminus10

MD

(f)


















4 Discussion




100

301

246

313

Control

lowastlowast

lowast

100

240 228

239 lowast

lowast



1

2

3

4

5

6

Fold

chan

ge

005

115

225

335

445

Fold

chan

ge

















5 Conclusion



Competing Interests




Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
































4 Discussion




100

301

246

313

Control

lowastlowast

lowast

100

240 228

239 lowast

lowast



1

2

3

4

5

6

Fold

chan

ge

005

115

225

335

445

Fold

chan

ge

















5 Conclusion



Competing Interests




Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















100

301

246

313

Control

lowastlowast

lowast

100

240 228

239 lowast

lowast



1

2

3

4

5

6

Fold

chan

ge

005

115

225

335

445

Fold

chan

ge

















5 Conclusion



Competing Interests




Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























5 Conclusion



Competing Interests




Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article potential osteoinductive effects of

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