research article modulation of dental pulp stem...

Research ArticleModulation of Dental Pulp Stem Cell Odontogenesis ina Tunable PEG-Fibrinogen Hydrogel System

Qiqi Lu1 Mirali Pandya1 Abdul Jalil Rufaihah2 Vinicius Rosa1 Huei Jinn Tong1

Dror Seliktar34 and Wei Seong Toh15

1Faculty of Dentistry National University of Singapore 11 Lower Kent Ridge Road Singapore 1190832Department of Surgery Yong Loo Lin School of Medicine National University Health System National University of Singapore1E Kent Ridge Road Singapore 1192883Nanoscience and Nanotechnology Initiative Faculty of Engineering National University of Singapore2 Engineering Drive 3 Singapore 1175814Faculty of Biomedical Engineering Technion-Israel Institute of Technology 32000 Haifa Israel5Tissue Engineering Program Life Sciences Institute National University of Singapore 27 Medical Drive Singapore 117510

Correspondence should be addressed to Wei Seong Toh dentohwsnusedusg

Received 22 December 2014 Revised 17 February 2015 Accepted 22 February 2015

Academic Editor Hai-Quan Mao

Copyright copy 2015 Qiqi Lu et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Injectable hydrogels have the great potential for clinical translation of dental pulp regeneration A recently developed PEG-fibrinogen (PF) hydrogel which comprises a bioactive fibrinogen backbone conjugated to polyethylene glycol (PEG) side chainscan be cross-linked after injection by photopolymerization The objective of this study was to investigate the use of this hydrogelwhich allows tuning of its mechanical properties as a scaffold for dental pulp tissue engineering The cross-linking degree ofPF hydrogels could be controlled by varying the amounts of PEG-diacrylate (PEG-DA) cross-linker PF hydrogels are generallycytocompatible with the encapsulated dental pulp stem cells (DPSCs) yielding gt85 cell viability in all hydrogels It was foundthat the cell morphology of encapsulated DPSCs odontogenic gene expression and mineralization were strongly modulated bythe hydrogel cross-linking degree andmatrix stiffness Notably DPSCs cultured within the highest cross-linked hydrogel remainedmostly rounded in aggregates anddemonstrated the greatest enhancement in odontogenic gene expressionConsistently the highestdegree of mineralization was observed in the highest cross-linked hydrogel Collectively our results indicate that PF hydrogels canbe used as a scaffold for DPSCs and offers the possibility of influencing DPSCs in ways that may be beneficial for applications inregenerative endodontics

1 Introduction

In recent years there has been an increasing focus on conser-vative strategies in endodontics for the treatment of diseaseddental pulps In the emerging paradigm of regenerative endo-dontics stem cell and tissue engineering technologies offerpotential for repair and regeneration of dentin-pulp tissuesto treat necrotic teeth in patients [1 2]

Dental pulp stem cells (DPSCs) have been demonstratedas a suitable cell source for dental tissue regeneration owingto their high accessibility proliferative ability and multilin-eage differentiation potential [3 4] Inductive biochemical

factors for odontoblastic differentiation of DPSCs have beenwell studied in recent years [5ndash7] However there is still lim-ited understanding of the ideal scaffold design and the criticalstem-cell biomaterial interactions that are needed to supportDPSCs to regenerate dental tissues Of note the mechanicalproperties of the artificial extracellular microenvironmentandhow theymay affect the behavior ofDPSCs are still poorlyunderstood

In the context of dental pulp tissue engineering the useof injectable hydrogels as scaffolds is particularly attractiveas they are expected to conform to the variable shape of thepulp chamber and can be formulatedwith cells andor growth

Hindawi Publishing CorporationStem Cells InternationalVolume 2015 Article ID 525367 9 pageshttpdxdoiorg1011552015525367

2 Stem Cells International

factors by simple mixing [8ndash12] We have previously devel-oped an injectable semisynthetic hydrogel scaffold materialcomprised of a fibrinogen backbone covalently conjugatedto polyethylene glycol (PEG) side chains and cross-linked byphotopolymerization The hydrogelrsquos mechanical propertiesmay also be tuned by the degree of cross-linking through asimple adjustment of the concentration of additional PEG-diacrylate (PEG-DA) photoinitiator and ultraviolet (UV)light intensity [13] Furthermore the unique photo-cross-linking property of the hydrogel would allow dentists to injectthe precursor solution into the dental pulp chamber beforerapid gelation by photopolymerization

In this study we examined the cytocompatibility ofPEG-fibrinogen (PF) hydrogels and the effects of varyingthe hydrogel cross-linking degrees on the differentiation ofencapsulated dental pulp stem cells (DPSCs) We hypothe-sized that hydrogels of varying cross-linking degrees mightinduce cellular morphological changes and impact on theextent of differentiationWe also hypothesized that PF hydro-gels with a higher cross-linking degree and correspondinglyhigher storage modulus would facilitate odontoblastic differ-entiation of DPSCsThis study may prove useful in designinginjectable hydrogel scaffolds for applications in dental pulptissue engineering and regenerative endodontics

2 Materials and Methods

21 Synthesis of PEG-Fibrinogen Precursors PEG fibrinogen(PF) was synthesized according to published protocols [13]Briefly 7mgmL bovine fibrinogen (Bovogen Biologicals PtyLtd Australia) was dissolved in 10mM phosphate bufferedsaline (PBS) with 8M urea Tris(2-carboxyethyl)phosphinehydrochloride (TCEP-HCL) was added to the solution ata molar ratio of 15 1 to the amount of cysteines in fib-rinogen The fibrinogen solution was then adjusted to pH 8using sodium hydroxide (NaOH)The PEG-DA solution wasprepared by dissolving 280mgmL PEG-DA (linear 10 kDa)powder in 10mM PBS with 8M urea After centrifugationthe supernatant containing dissolved PEG-DA was addedto the fibrinogen solution at a 37 1 molar ratio of PEGto fibrinogen cysteines The mixture was then reacted in areaction vessel with a thermostatic jacket (Lenz LaborglasGermany) at 225∘C for 3 h in the dark After reactionthe solution was first diluted with equal amount of PBScontaining 8M urea and then precipitated by adding intoacetone at a volume ratio of 4 1 (acetone to solution) Theprecipitant was redissolved by adding 18 volumes of PBS-8M urea The modified fibrinogen was then purified andconcentrated to 8ndash12mgmL by using Centramate cassettes(50 kDaMW cutoff NY USA) The PEG-fibrinogen (PF)solution was subsequently passed through a high shear fluidprocessor (Microfluidics M110-P USA) to achieve uniformparticle size and finally filtered with VacuCap 90 filter (PallCorporation USA)

22 Fabrication of PF Hydrogels PF hydrogels with differentcross-linking degrees were made by cross-linking PF gelsusing variable amount of additional PEG-DA Four groups

containing 0 05 15 and 25 wv additional PEG-DA wereprepared To make these gels a precursor solution contain-ing four components was prepared (1) PF hydrogel (finalconcentration 8mgmL) (2) PEG-DA (diluted from 15stock solution in PBS) (3) 1 (vv) photoinitiator solution(prepared from stock solution containing 10 wv Irgacure2959 (Ciba Switzerland) in 70 ethanol) (4) PBS (volumeadjustable) The precursor solutions were first mixed byvortexing for 3 s and then transferred to plastic cylindermolds (150 120583L Fisher Scientific) or 15-well 120583-slides (Ibidi) forgel casting To initiate cross-linking the precursor solutionswere exposed to long-wave UV light (365 nm 4-5mWcm2)for 3min in 15-well 120583-slide or 5min in plastic cylinder molds

23 Rheological Characterization of PF Hydrogels Rheolog-ical measurements of PF hydrogels were carried out usingAR-G2 rheometer (TA instruments New Castle DE USA)according to a published protocol [14] Briefly after 1minequilibrium time 200120583L PF precursor solution loaded on theinstrument was cross-linked by exposure to long-wave UVlight (365 nm) emitted from OmniCure Series 2000 UV lightsource at an intensity of 5mWcm2 Time-sweep oscillatorytests were done at room temperature at a sinusoidal 2 strainrate and a 6-rad sminus1 angular frequencywithin the linear visco-elastic region as determined previously [15] Temporal changeof storage modulus (1198661015840) was recorded for 3min after initia-tion of cross-linking

24 Swelling Analysis of PF Hydrogels For measuring theswelling ratio PF hydrogels were cast into cylinder moldstransferred to PBS and allowed to equilibrate for 24 h at 4∘CWet weight of the samples was determined before undergo-ing freeze-drying overnight The change in hydrogel weightbetween the swollen (119882

119904) and dried (119882

119889) states was used to

determine the volumetric swelling ratio as follows

Swelling ratio =119882119904

119882119889

(1)

25 DPSC Encapsulation and Differentiation Human DPSCsused in this study were obtained commercially fromAllCellsLLC DPSCs were cultured in Dulbeccorsquos modified eaglersquosmedium (DMEM) (low glucose Biowest France) supple-mented with 10 fetal bovine serum (FBS Biowest) and 1penicillinstreptomycin (PS Life technologies Singapore) Toinitiate cell encapsulation DPSCs at P4-P5 were detached bytrypsinization before being resuspended in PF gel precursorsolution at a density of 1 times 106 cellsmL To initiate cross-linking the cell-gel mixtures were exposed to long-waveUV light for 3min in 15-well 120583-slide or 5min in plasticcylindermoldsDPSCs encapsulated in the PFhydrogelsweredifferentiated to odontoblast-like cells by culturing in odon-togenicosteogenic differentiation medium [16] comprised ofDMEMhigh glucose 10 FBS 1 PS 10minus7Mdexamethasone(Sigma St Louis MO USA) 50120583gmL ascorbic acid 2-phosphate (Sigma) and 10mM 120573-glycerophosphate (Sigma)DPSCs encapsulated in PF hydrogels (cast in both cylindermolds and in 15-well 120583-slides) were differentiated for 3 weekswith medium change every 3 days

Stem Cells International 3

Table 1 List of primers

Target Forward ReverseCOL I AAAAGGAAGCTTGGTCCACT GTGTGGAGAAAGGAGCAGAADSPP TTAAATGCCAGTGGAACCAT ATTCCCTTCTCCCTTGTGACDMP-1 TGGGGATTATCCTGTGCTCT TACTTCTGGGGTCACTGTCGOC CATGAGAGCCCTCACA AGAGCGACACCCTAGACGAPDH GAGTCAACGGATTTGGTCGT GACAAGCTTCCCGTTCTGAG

26 Cell Viability Encapsulated DPSCs in PF hydrogels werestained by livedead viability assay kit (Life Technologies)according to themanufacturerrsquos protocol Briefly themediumwas removed and the cells in hydrogel were rinsed oncewith PBS followed by incubation with the dye Live cellsstained green with calcein AM and dead cells marked redwith ethidium homodimer-1 (EthD-1) were visualized usinga confocal microscope Two to three random sections wereanalyzed for each sample (119899 = 2-3 samples per gel type)The cell count was performed using ImageJ software (NIHBethesda MD) Percentage cellular viability was calculatedas the number of viable cells is divided by the total numberof cells measured per gel multiplied by 100

27 Cell Morphology After 21 days of culture DPSC-ladenhydrogel constructs were fixed in 10 buffered formalinovernight at 4∘C The PF hydrogel constructs were furthercryoprotected in 25 sucrose solution flash-frozen in isobu-tane (minus30∘C) embedded in Tissue-Tek OCT compound(Sakura Finetek Inc Torrance CA USA) and cryosectionedat 30 120583m [17] The sections were stained with haematoxylinand eosin (HampE) following the standard histology techniqueas previously described [18] Cellmorphologywas assessed byanalysis of the HampE stained cross-sections of the PF hydrogelconstructs Digital micrographs were taken at 10x objectivelens magnification and percentage of cellular aggregationwas analyzed using ImageJ software (NIH Bethesda MD)and expressed as the percentage of total number of cells aspreviously described [19] Two to three random sections wereanalyzed for each sample (119899 = 2-3 samples per gel type)

28 Real-Time RT-PCR For real-time reverse transcriptasepolymerase chain reaction (RT-PCR) PF hydrogels werecasted in cylinder molds with 1 times 106mL DPSCs TotalRNA of DPSCs was extracted by NucleoSpin RNA extractionkit (Macherey-Nagel Germany) Purified RNA was thentranscribed to cDNA by iScript cDNA synthesis kit (BioradHercules CA USA) For PCR reaction 2X iScript one-stepRT-PCR reagent (Biorad) was mixed with cDNA templatesand primers The RT-PCR reactions were performed in CFXConnect real-time PCR machine (Biorad) at 95∘C for 3minfollowed by 40 cycles of 10 s denaturation at 95∘C and 30 sannealing at 55∘C For all experiments glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as internalreference gene and 3 samples (119899 = 3 samples per gel type)were assigned in each group for quantification The primersequences of collagen I (Col I) dentin sialophosphoprotein

(DSPP) dentin matrix protein-1 (DMP-1) osteocalcin (OC)and GAPDH are detailed in Table 1

29 Calcium Assay and DNA Quantification Calcium assayand DNA quantification required lysis of PF hydrogels andthe encapsulated DPSCs After differentiation for 3 weekssamples were transferred to 15mL microtubes containingPBS with 02 Tween 20 (Sigma) for lysis After incubationat 37∘C for 2 h the lysed samples were stored at minus20∘C forsubsequent assays For measurements of calcium and DNAamounts calcium assay (Cayman Ann Arbor MI USA) andPicoGreen dsDNA quantification assay (Life Technologies)were performed respectively according to the manufactur-ersrsquo instructions Readings were taken using the Infinite 2000plate reader (Tecan Austria) The amount of calcium presentin each sample was normalized to the corresponding DNAamount

210 Statistical Analysis The data were reported as meanplusmn standard deviation (SD) Analysis of variance (ANOVA)and Fisherrsquos protected least squares difference (PLSD) posthoc testing were performed using StatView software (SASInstitute Inc Cary NC USA) Statistical significance was setas 119875 lt 005

3 Results

31 Hydrogel Characterization The amount of additionalPEG-DA cross-linker (from 0 to 25 wt ) was varied to formPEG-fibrinogen (PF) hydrogels of four different cross-linkingdegrees (PF-0 PF-05 PF-15 and PF-25) respectively whichspanned a range of mechanical and swelling properties

The formation of PF hydrogels was evaluated using oscil-latory rheometry which measures the shear storage modulus(1198661015840

) and loss modulus (11986610158401015840) against the shear strain The gelpoint defined as the crossover of1198661015840 and11986610158401015840 was employed toevaluate the gelation rate of the hydrogel As summarized inTable 2 the peak values of 1198661015840 that represented the stiffness ofthe fully cross-linked hydrogel were tunable by the amount ofPEG-DA added to the precursor solutionsThe addition of 005 15 and 25 wtPEG-DA resulted inmean peak1198661015840 valuesof 140plusmn 2 454plusmn 13 1574plusmn 21 and 3601plusmn 47Pa respectivelyThis change in1198661015840with increasingPEG-DApercentage provedto be statistically significant (119875 lt 00001 power = 1) Alsothe gel point of the hydrogels ranged from 114 to 234 s withincreasing amount of PEG-DA In addition the time requiredfor 1198661015840 to reach the peak and then plateau also increased


Table 2 Characterization of PEG-fibrinogen (PF) hydrogelsa

Gel type PEG-DA 1198661015840 (Pa) Gel point (s)b Time required to reach 1198661015840 plateau (s)

PF-0 0 1401 plusmn 18 114 plusmn 02 382 plusmn 02PF-05 05 4538 plusmn 126 115 plusmn 01 517 plusmn 01PF-15 15 1574 plusmn 212 174 plusmn 26 826 plusmn 01PF-25 25 3601 plusmn 465 234 plusmn 25 1274 plusmn 45aMeasurements were taken at room temperature in the time-sweep oscillatory mode with a sinusoidal 2 strain rate and a 6 rad sminus1 angular frequency (Meanplusmn SD 119899 = 3)bGel point is defined as the time at which the crossover of storage modulus (1198661015840) and loss modulus (11986610158401015840) occurred Herein it is used as an indicator of the rateof gelation

0

20

40

60

PF-0 PF-05 PF-15 PF-25

Swel

ling

ratio

lowastlowastlowast

lowast

Figure 1 Swelling ratio of PF hydrogels (PF-0 PF-05 PF-15and PF-25) cross-linked with 0 05 15 and 25 of PEG-DArespectively Mean plusmn SD 119899 = 3group Analysis revealed significanteffects of cross-linking by the addition of PEG-DA on the swellingratio (lowast119875 lt 005)

significantly with the increase in PEG-DA percentage (119875 lt00001 power = 1) These results indicate that the additionof PEG-DA cross-linker allows for a higher degree of PFhydrogel cross-linking that results in higher modulus butalso requires longer times for completion of the cross-linkingprocess

The addition of PEG-DA cross-linker significantlydecreased the swelling ratio (119875 lt 005 power = 071)as shown in Figure 1 Notably the swelling ratio decreasedfrom 42 plusmn 75 in PF-0 to 293 plusmn 08 in PF-25 hydrogelThese results indicate that an inverse correlation existsbetween the amount of additional PEG-DA and the swell-ing ratio whereby hydrogels with higher PEG-DA concentra-tion induce less swelling due to higher network cross-linkingdegree with a smaller mesh size

32 DPSC Viability and Cell Shape Changes in PF HydrogelsDPSCs were encapsulated within PF hydrogels (PF-0 PF-05 PF-15 and PF-25) during photopolymerization to create3D cell-seeded constructs Cellular viability was assessedusing livedead staining after 1 day and 7 days in culture(Figure 2(a)) Viability assessed by livedead staining 1 dayafter encapsulation indicated high viability (gt90) ofDPSCsencapsulated in PF-0 with an observable trend of decreas-ing cellular viability with increasing percentage of PEG-DAcross-linker Approximately 15 cell death was observed in

PF-25 hydrogels (Figure 2(b)) By day 7 of culture greaterthan 85 viability was observed in all hydrogels althoughthere seemed to have a slight decrease in cell number possiblydue to cell aggregation

In PF-0 and PF-05 hydrogels cells were initially roundedbut rapidly became spindled within 24 h (Figure 2(a)) By day7 in culture most of the DPSCs became highly spindled Asmall number of cell clusters with spindle-like cytoplasmicextensions could also be observed Notably DPSCs remainedspindled in PF-0 and PF-05 hydrogels for the duration oftime in culture (Figure 3) Conversely in PF-15 and PF-25 hydrogels DPSCs exhibited fewer cell extensions and adecreased spindled morphology Evidently as revealed in thehistological cross-sections of the gel constructs cells wereless able to form extensions in the dense polymer networkcontaining the additional PEG-DA cross-linker (PF-15 andPF-25) and remained mostly rounded in aggregates for theduration of 21-day culture (Figure 3(a)) Quantitative analysisfurther indicated a relationship between the degree of cross-linking and the extent of cell aggregation (Figure 3(b)) Bythe end of 21-day culture the highest cross-linked PF-25hydrogels exhibited the highest percentage of cell aggregation(886 plusmn 98) With increase in gel cross-linking there wasa significant increase in percentage of cell aggregates (Fig-ure 3(b)) One-factor ANOVA revealed significant effect ofgel cross-linking on the extent of cell aggregation (119875 lt00001 power = 1)

33 DPSC Differentiation andMineralization in PF HydrogelsThe effect of the mechanical properties of the tunable 3D PFhydrogels on the differentiation of DPSCs under odontogenicconditions over a 21-day time course was assessed (Fig-ure 4) The expression levels of genes related to the odonto-genic differentiation ofDPSCs includingCol IDSPPDMP-1andOC were measured using quantitative real-time RT-PCRat days 7 and 21 of differentiation (Figure 4) The expressionlevel of Col I gene was highest in PF-0 hydrogels at day 7of differentiation and increased with culture time By day21 of differentiation gene expression level of Col I in PF-0 hydrogel was approximately 13-fold higher than that inPF-25 hydrogel (Figure 4(a) 119875 lt 0001) By contrast theexpression level of Col I gene was consistently the lowestin PF-25 hydrogels at different time points (Figure 4(a))Therewere no significant differences in gene expression levelsof DSPP and DMP-1 among the PF hydrogels at day 7 of


Day 1 Day 7PF

-0PF

-05

PF-1

5PF

-25

Scale bar = 200120583m(a)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

Day 1Day 7

PF-0 PF-05 PF-15 PF-25

lowast

(b)Figure 2 Cytocompatibility of PF hydrogels (PF-0 PF-05 PF-15 and PF-25) as a function of degree of cross-linking after 1 and 7 days ofculture (a) Representative livedead images of DPSC-laden PF hydrogels at days 1 and 7 of culture with live cells stained green and dead cellsshown in red (119899 = 3grouptime point scale bar = 200120583m) (b) Percentage of cell viability of DPSCs at days 1 and 7 of culture Mean plusmn SD119899 = 3 2-3 imagesgel sample lowast119875 lt 005 Fisherrsquos post hoc test

PF-0 PF-05

PF-15 PF-25


0

20

40

60

80

100

120

PF-0 PF-05 PF-15 PF-25

Cell

aggr

egat

ion

()

lowastlowastlowast

(b)Figure 3 Effect of hydrogel degree of cross-linking on morphology of encapsulated DPSCs (a) Representative images of DPSC-laden PFhydrogels following 21 days of culture (119899 = 3 scale bar = 100120583m) (b) Percentage of cell aggregation after 21 days of culture Mean plusmn SD 119899 = 32-3 imagesgel sample lowastlowastlowast119875 lt 0001 Fisherrsquos post hoc test


Col I

1

10

100

7 21

Relat

ive g

ene e

xpre

ssio

n

PF-0PF-05

PF-15PF-25

Days

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

lowastlowastlowast

lowastlowastlowast

(a)

DSPP

PF-0PF-05

PF-15PF-25

1

10

100

1000

10000

100000

7 21

Relat

ive g

ene e

xpre

ssio

n

Days

lowastlowastlowast

(b)

DMP-1

PF-0PF-05

PF-15PF-25

1

10

100

1000

7 21

Relat

ive g

ene e

xpre

ssio

n

Days

lowastlowastlowast

(c)

OC

1

10

100

7 21

Relat

ive g

ene e

xpre

ssio

n

PF-0PF-05

PF-15PF-25

Days

lowastlowastlowast

lowastlowast

lowast

lowast

(d)

Figure 4 Odontogenic differentiation of encapsulated DPSCs in PF hydrogels (PF-0 PF-05 PF-15 and PF-25) as a function of degree ofcross-linking and culture time Relative gene expression levels of Col I DSPP DMP-1 and OC were determined with respect to the day 0expression level and presented as bar graphs using logarithmic scales Mean plusmn SD 119899 = 3grouptime point lowast119875 lt 005 lowastlowast119875 lt 001 andlowastlowastlowast

119875 lt 0001 Fisherrsquos post hoc test

differentiation (Figures 4(b) and 4(c)) However by day 21of differentiation the gene expression levels of DSPP andDMP-1 increased in PF hydrogels with higher PEG-DAand were the highest in PF-25 hydrogels (Figures 4(b) and4(c)) Notably gene expression levels of DSPP and DMP-1 inPF-25 hydrogels were approximately 1800-fold (119875 lt 0001)and 150-fold (119875 lt 0001) higher than that in PF-0 hydrogelsrespectively The gene expression levels of OC were higherin PF hydrogels with added PEG-DA (05 15 and 25) atday 7 of differentiation (Figure 4(d)) However by day 21 ofdifferentiation the gene expression levels ofOC in PF-05 andPF-15 hydrogels decreased to basal levels comparable to that

in PF-0 hydrogel while gene expression level of OC in PF-25 increased with culture time Notably the gene expressionof OC in PF-25 hydrogel was at least 5-fold higher than theother PF hydrogels (Figure 4(d) 119875 lt 0001)

The mineralization in hydrogel scaffolds was exam-ined using Alizarin red staining and calcium quantitativeassay (Figure 5) Calcium quantification revealed signifi-cantly higher levels of calcium deposition in PF hydrogelswith higher PEG-DA (Figure 5(a)) One-factor ANOVArevealed significant effect of gel cross-linking on the extent ofcalcium deposition (119875 lt 00001 power = 1) By the end of 21-day differentiation the DNA content (number of DPSCs) was


0

20

40

60

80

PF-0 PF-05 PF-15 PF-25

lowastlowastlowast

lowastlowast

lowast

(120583g

calc

ium

gel

cons

truc

t)

(a)

0

20

40

60

80

100

PF-0 PF-05 PF-15 PF-25

lowastlowastlowast

lowastlowast

lowastlowast

lowast

(ng

DN

Ag

el co

nstr

uct)

(b)

0

06

12

18

PF-0 PF-05 PF-15 PF-25

lowastlowastlowast

(120583g

calc

ium

ng

DN

A)

(c)

PF-0 PF-05

PF-15 PF-25

Aliz

arin

red

Scale bar = 500120583m

(d)

Figure 5 Calcium quantification of encapsulated DPSCs in PF hydrogels (PF-0 PF-05 PF-15 and PF-25) as a function of degree of cross-linking after 21 days of culture (a) Total calcium content per gel construct (b) DNA content per gel construct (c) Calcium content per cellbased on the amount of calcium normalized by the DNA content in the construct Mean plusmn SD 119899 = 3 (d) Alizarin red staining of the DPSC-laden gel constructs Red color indicates calcium deposition Scale bar = 500 120583m lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 Fisherrsquos post hoctest

the lowest in the highest cross-linked PF-25 hydrogel (Fig-ure 5(b)) When normalized by the DNA content highestlevel of calcium content on a per cell basis was observed inPF-25 hydrogel with a value of at least 35-fold higher thanthe values for the other PF hydrogels (Figure 5(c)119875 lt 0001)This result was further confirmed by Alizarin red staining(Figure 5(d)) that observed mineralization only in PF-15 andPF-25 hydrogels where cells remained mostly rounded inaggregates In contrast no mineralization was observed inPF-0 and PF-05 where most cells remained spindled

4 Discussion

Theuse of injectable and in situ gel-forming systems is partic-ularly attractive for clinical translation of dental pulp regen-eration as they can be easily formulated with growth factorsand cells by simple mixing and can conform to the variableshape of the pulp chamber following injection Thereforethere has been a surge of interest in the development of

injectable hydrogels in recent years for tissue engineering[20] particularly dental pulp tissue engineering [8ndash12]

In the design of the scaffold for dental pulp tissueengineering several parameters including the matrix com-position and architecture have been reported to influenceadhesion proliferation and differentiation of dental stemcells and progenitors While most studies [11 12 16] focuson modification of the scaffold to enhance odontogenicdifferentiation and biomineralization the influence of matrixstiffness on the differentiation ofDPSCs is still largely unclearSeparately several studies have reported the influence ofmatrix stiffness on modulation of cell fate of a wide varietyof cell types including stem cells [21ndash26] In particular in alandmark study Engler et al demonstrated that the elasticityof the matrix influences the differentiation of MSCs intoneurons myoblasts and osteoblasts in ascending order ofstiffness with the stiffest matrices supporting MSC differen-tiation to osteoblasts [21] Thus it is the goal of this studyto investigate the effects of varying hydrogel cross-linking


degrees and corresponding matrix stiffness on the differen-tiation of DPSCs using a tunable hydrogel system

Previous studies utilizing natural biopolymer gels (col-lagen Matrigel PuraMatrix and hyaluronic acid) do notprovide a means to tune the mechanical properties indepen-dently from matrix composition that confers biofunctionalproperties of adhesion and proteolytic sensitivity [8 9 12]The semisynthetic PEG-fibrinogen (PF) hydrogel was partic-ularly useful for this study because the mechanical propertiesare tunable by the addition of cross-linker (PEG-DA) thatcontrols the hydrogel cross-linking degree whilemaintaininga constant fibrinogen backboneThis unique feature allows usto investigate the effects of mechanical properties of hydrogelon DPSC differentiation systematically and independently

In general PF hydrogels were cytocompatible withDPSCs although there was a slight decrease in cell viabilitywith increasing cross-linking degree and matrix stiffnessDistinct differences in cell morphology of DPSCs could beobserved in PF hydrogels with varying cross-linking degreesEvidently cells formed clusters with spindle-like cytoplasmicextensions within the lower cross-linked PF hydrogels (PF-0 and PF-05) which is frequently observed as a commonfeature of these cells in soft matrices such as the PuraMatrix[8 9] The ability to spread and elongate may also relateto the ability of the cells to secrete fibrinolytic enzymesto break apart the hydrogel network more readily in thelower cross-linked hydrogels than in the higher cross-linkedcounterparts [27] Conversely cells tended to be roundedand forming aggregates confined within the dense polymernetwork of the higher cross-linked hydrogels (PF-15 and PF-25) It is well known that cell morphology and functionare tightly coupled [28] Notably lower cross-linked matricesthat induced spindle-like elongation of DPSCs resulted inmore pronounced gene expression of Col I Although Col Iis one of the extracellular matrix (ECM) components of thedemineralized dentin it is also a major collagenous ECMprotein present in many other tissues With the concomitantupregulation in Col I but downregulation of other odon-togenic markers including DSPP DMP-1 and OC there islikelihood that DPSCs were guided by the softer matricesto differentiate to lineages other than the odontoblasts andosteoblasts

On the other hand the higher cross-linked stiffer hydro-gels (PF-15 and PF-25) tested in this study with a1198661015840 rangingfrom 1500 to 3600 Pa generally favored odontoblastic dif-ferentiation of DPSCs The PF-25 hydrogel constructs at astiffness of 1198661015840 sim3600 Pa induced the highest gene expressionofDSPPDMP-1 andOC by the end of 21-day differentiationIt is likely that the heightened odontogenicosteogenic dif-ferentiation is a result of the DPSCs forming cell aggregatesandmechanosensing the stiffermatrix that has been shown inprevious studies to promote stem cell osteogenesis [29 30]Consistent with the gene expression results mineralizationwas only observed in higher cross-linked PF-15 and PF-25 hydrogels with the latter showing the most robustmineralization Of interest there was a strong correlationbetween cellular aggregation denoted by the percentage ofrounded cells and the extent of calcium deposition (linearregression analysis 1198772 gt 092)

With the lowest relative cell number by the end of 21-daydifferentiation it is likely that cellular aggregation observedin the highest cross-linked PF-25 hydrogel promoted odon-togenic differentiation but lesser extent of proliferationNevertheless a more thorough and quantitative assessmentwould be necessary for a more complete characterization ofthe cellular proliferation during the course of differentiation

Collectively the injectable PF hydrogels are cytocompati-ble with DPSCs and the hydrogel mechanical properties (iecross-linking degree and matrix stiffness) and biofunctionalproperties conferred by fibrinogen could be tuned to pro-vide a supportive biomimetic cellular microenvironment forodontogenic differentiation These results suggest a possibleuse of these hydrogels as scaffolds for dentin-pulp tissueengineering and regeneration To the best of our knowledgethis work represents the first demonstration of the influenceofmatrix stiffness onDPSCodontogenic differentiation in 3Dtunable hydrogels Further studies through tooth slice organculture [31] and in vivo transplantation studies [9 32] wouldbe needed to assess if these hydrogels are supportive of theformation of new tubular dentin and pulp tissue complex fordental pulp regeneration

5 Conclusions

Injectable in situ forming hydrogels are particularly attractivefor dental pulp tissue engineering as they can be easilyformulated with growth factors and cells by simple mixingand can conform to the pulp chamber following injection Inthis study we investigated the use of injectable PF hydrogelsas scaffold carriers for DPSCs for dental pulp tissue engi-neering and provided strong evidence that the tunable 3Dmicroenvironment of the PF-hydrogels modulates odonto-genic differentiation and mineralization of human DPSCs

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by National Research Foundation-(NRF-) Technion-NUS grant and grants (R221000067133R221000068720 and R221000070733) from National Uni-versity of Singapore (NUS) National University HealthcareSystem and Ministry of Education Singapore

References

[1] V Rosa A Della Bona B N Cavalcanti and J E Nor ldquoTissueengineering from research to dental clinicsrdquo Dental Materialsvol 28 no 4 pp 341ndash348 2012

[2] V Rosa T M Botero and J E Nor ldquoRegenerative endodonticsin light of the stem cell paradigmrdquo International Dental Journalvol 61 no 1 pp 23ndash28 2011

[3] G T-JHuang SGronthos and S Shi ldquoMesenchymal stemcellsderived from dental tissues vs those from other sources theirbiology and role in regenerative medicinerdquo Journal of DentalResearch vol 88 no 9 pp 792ndash806 2009


[4] G T-J Huang K Shagramanova and S W Chan ldquoFormationof odontoblast-Like cells from cultured human dental pulp cellson dentin In Vitrordquo Journal of Endodontics vol 32 no 11 pp1066ndash1073 2006

[5] Y-S KimK-SMinD-H Jeong J-H JangH-WKim andE-C Kim ldquoEffects of fibroblast growth factor-2 on the expressionand regulation of chemokines in human dental pulp cellsrdquoJournal of Endodontics vol 36 no 11 pp 1824ndash1830 2010

[6] L Karanxha S-J Park W-J Son J E Nor and K-S MinldquoCombined effects of simvastatin and enamel matrix derivativeon odontoblastic differentiation of human dental pulp cellsrdquoJournal of Endodontics vol 39 no 1 pp 76ndash82 2013

[7] X Yang P M van der Kraan J V D Dolder et al ldquoSTRO-1selected rat dental pulp stem cells transfected with adenoviral-mediated human bone morphogenetic protein 2 gene showenhanced odontogenic differentiationrdquo Tissue Engineering vol13 no 11 pp 2803ndash2812 2007

[8] B N Cavalcanti B D Zeitlin and J E Nor ldquoA hydrogel scaffoldthat maintains viability and supports differentiation of dentalpulp stem cellsrdquoDentalMaterials vol 29 no 1 pp 97ndash102 2013

[9] V Rosa Z Zhang R H M Grande and J E Nor ldquoDental pulptissue engineering in full-length human root canalsrdquo Journal ofDental Research vol 92 no 11 pp 970ndash975 2013

[10] K M Galler J D Hartgerink A C Cavender G Schmalz andR N DrsquoSouza ldquoA customized self-assembling peptide hydrogelfor dental pulp tissue engineeringrdquo Tissue EngineeringmdashPart Avol 18 no 1-2 pp 176ndash184 2012

[11] S-J Park Z Li I-N Hwang KMHuh and K-SMin ldquoGlycolchitin-based thermoresponsive hydrogel scaffold supplementedwith enamel matrix derivative promotes odontogenic differen-tiation of human dental pulp cellsrdquo Journal of Endodontics vol39 no 8 pp 1001ndash1007 2013

[12] L Lambricht Pa De Berdt J Vanacker et al ldquoThe typeand composition of alginate and hyaluronic-based hydrogelsinfluence the viability of stem cells of the apical papillardquo DentalMaterials vol 30 no 12 pp e349ndashe361 2014

[13] L Almany and D Seliktar ldquoBiosynthetic hydrogel scaffoldsmade from fibrinogen and polyethylene glycol for 3D cellculturesrdquo Biomaterials vol 26 no 15 pp 2467ndash2477 2005

[14] M Plotkin S R Vaibavi A J Rufaihah et al ldquoThe effect ofmatrix stiffness of injectable hydrogels on the preservation ofcardiac function after a heart attackrdquo Biomaterials vol 35 no5 pp 1429ndash1438 2014

[15] D Dikovsky H Bianco-Peled and D Seliktar ldquoDefining therole of matrix compliance and proteolysis in three-dimensionalcell spreading and remodelingrdquo Biophysical Journal vol 94 no7 pp 2914ndash2925 2008

[16] J Wang H Ma X Jin et al ldquoThe effect of scaffold architectureon odontogenic differentiation of human dental pulp stemcellsrdquo Biomaterials vol 32 no 31 pp 7822ndash7830 2011

[17] T C Lim S Rokkappanavar W S Toh L-S Wang M Kuri-sawa andM Spector ldquoChemotactic recruitment of adult neuralprogenitor cells into multifunctional hydrogels providing sus-tained SDF-1120572 release and compatible structural supportrdquo TheFASEB Journal vol 27 no 3 pp 1023ndash1033 2013

[18] W S Toh and T Cao ldquoDerivation of chondrogenic cells fromhuman embryonic stem cells for cartilage tissue engineeringrdquo inMethods inMolecular Biology Humana Press Totowa NJ USA2015

[19] I Martin B Dozin R Quarto R Cancedda and F BeltrameldquoComputer-based technique for cell aggregation analysis andcell aggregation in in vitro chondrogenesisrdquo Cytometry vol 28no 2 pp 141ndash146 1997

[20] W S Toh and X J Loh ldquoAdvances in hydrogel delivery systemsfor tissue regenerationrdquo Materials Science and Engineering Cvol 45 pp 690ndash697 2014

[21] A J Engler S Sen H L Sweeney and D E Discher ldquoMatrixelasticity directs stem cell lineage specificationrdquo Cell vol 126no 4 pp 677ndash689 2006

[22] S R Peyton P D Kim C M Ghajar D Seliktar and A JPutnam ldquoThe effects of matrix stiffness and RhoA on the phe-notypic plasticity of smooth muscle cells in a 3-D biosynthetichydrogel systemrdquo Biomaterials vol 29 no 17 pp 2597ndash26072008

[23] T C Lim W S Toh L-S Wang M Kurisawa and M SpectorldquoThe effect of injectable gelatin-hydroxyphenylpropionic acidhydrogel matrices on the proliferation migration differentia-tion and oxidative stress resistance of adult neural stem cellsrdquoBiomaterials vol 33 no 12 pp 3446ndash3455 2012

[24] W S Toh T C Lim M Kurisawa and M Spector ldquoModu-lation of mesenchymal stem cell chondrogenesis in a tunablehyaluronic acid hydrogel microenvironmentrdquo Biomaterials vol33 no 15 pp 3835ndash3845 2012

[25] L-S Wang C Du W S Toh A C A Wan S J Gao and MKurisawa ldquoModulation of chondrocyte functions and stiffness-dependent cartilage repair using an injectable enzymaticallycrosslinked hydrogelwith tunable mechanical propertiesrdquo Bio-materials vol 35 no 7 pp 2207ndash2217 2014

[26] D A Young Y S Choi A J Engler and K L ChristmanldquoStimulation of adipogenesis of adult adipose-derived stem cellsusing substrates that mimic the stiffness of adipose tissuerdquoBiomaterials vol 34 no 34 pp 8581ndash8588 2013

[27] D Dikovsky H Bianco-Peled and D Seliktar ldquoThe effect ofstructural alterations of PEG-fibrinogen hydrogel scaffolds on3-D cellular morphology and cellular migrationrdquo Biomaterialsvol 27 no 8 pp 1496ndash1506 2006

[28] M D Treiser E H Yang S Gordonov et al ldquoCytoskeleton-based forecasting of stem cell lineage fatesrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 107 no 2 pp 610ndash615 2010

[29] Y S Pek A C A Wan and J Y Ying ldquoThe effect of matrixstiffness on mesenchymal stem cell differentiation in a 3Dthixotropic gelrdquo Biomaterials vol 31 no 3 pp 385ndash391 2010

[30] J C Chen and C R Jacobs ldquoMechanically induced osteogeniclineage commitment of stem cellsrdquo Stem Cell Research amp Ther-apy vol 4 no 5 article 107 2013

[31] V T Sakai M M Cordeiro Z Dong Z Zhang B D Zeitlinand J E Nor ldquoTooth slicescaffold model of dental pulp tissueengineeringrdquo Advances in Dental Research vol 23 no 3 pp325ndash332 2011

[32] X Zhu C Zhang G T-J Huang G S P Cheung W L Dis-sanayaka and W Zhu ldquoTransplantation of dental pulp stemcells and platelet-rich plasma for pulp regenerationrdquo Journal ofEndodontics vol 38 no 12 pp 1604ndash1609 2012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


factors by simple mixing [8ndash12] We have previously devel-oped an injectable semisynthetic hydrogel scaffold materialcomprised of a fibrinogen backbone covalently conjugatedto polyethylene glycol (PEG) side chains and cross-linked byphotopolymerization The hydrogelrsquos mechanical propertiesmay also be tuned by the degree of cross-linking through asimple adjustment of the concentration of additional PEG-diacrylate (PEG-DA) photoinitiator and ultraviolet (UV)light intensity [13] Furthermore the unique photo-cross-linking property of the hydrogel would allow dentists to injectthe precursor solution into the dental pulp chamber beforerapid gelation by photopolymerization

In this study we examined the cytocompatibility ofPEG-fibrinogen (PF) hydrogels and the effects of varyingthe hydrogel cross-linking degrees on the differentiation ofencapsulated dental pulp stem cells (DPSCs) We hypothe-sized that hydrogels of varying cross-linking degrees mightinduce cellular morphological changes and impact on theextent of differentiationWe also hypothesized that PF hydro-gels with a higher cross-linking degree and correspondinglyhigher storage modulus would facilitate odontoblastic differ-entiation of DPSCsThis study may prove useful in designinginjectable hydrogel scaffolds for applications in dental pulptissue engineering and regenerative endodontics

2 Materials and Methods

21 Synthesis of PEG-Fibrinogen Precursors PEG fibrinogen(PF) was synthesized according to published protocols [13]Briefly 7mgmL bovine fibrinogen (Bovogen Biologicals PtyLtd Australia) was dissolved in 10mM phosphate bufferedsaline (PBS) with 8M urea Tris(2-carboxyethyl)phosphinehydrochloride (TCEP-HCL) was added to the solution ata molar ratio of 15 1 to the amount of cysteines in fib-rinogen The fibrinogen solution was then adjusted to pH 8using sodium hydroxide (NaOH)The PEG-DA solution wasprepared by dissolving 280mgmL PEG-DA (linear 10 kDa)powder in 10mM PBS with 8M urea After centrifugationthe supernatant containing dissolved PEG-DA was addedto the fibrinogen solution at a 37 1 molar ratio of PEGto fibrinogen cysteines The mixture was then reacted in areaction vessel with a thermostatic jacket (Lenz LaborglasGermany) at 225∘C for 3 h in the dark After reactionthe solution was first diluted with equal amount of PBScontaining 8M urea and then precipitated by adding intoacetone at a volume ratio of 4 1 (acetone to solution) Theprecipitant was redissolved by adding 18 volumes of PBS-8M urea The modified fibrinogen was then purified andconcentrated to 8ndash12mgmL by using Centramate cassettes(50 kDaMW cutoff NY USA) The PEG-fibrinogen (PF)solution was subsequently passed through a high shear fluidprocessor (Microfluidics M110-P USA) to achieve uniformparticle size and finally filtered with VacuCap 90 filter (PallCorporation USA)

22 Fabrication of PF Hydrogels PF hydrogels with differentcross-linking degrees were made by cross-linking PF gelsusing variable amount of additional PEG-DA Four groups

containing 0 05 15 and 25 wv additional PEG-DA wereprepared To make these gels a precursor solution contain-ing four components was prepared (1) PF hydrogel (finalconcentration 8mgmL) (2) PEG-DA (diluted from 15stock solution in PBS) (3) 1 (vv) photoinitiator solution(prepared from stock solution containing 10 wv Irgacure2959 (Ciba Switzerland) in 70 ethanol) (4) PBS (volumeadjustable) The precursor solutions were first mixed byvortexing for 3 s and then transferred to plastic cylindermolds (150 120583L Fisher Scientific) or 15-well 120583-slides (Ibidi) forgel casting To initiate cross-linking the precursor solutionswere exposed to long-wave UV light (365 nm 4-5mWcm2)for 3min in 15-well 120583-slide or 5min in plastic cylinder molds

23 Rheological Characterization of PF Hydrogels Rheolog-ical measurements of PF hydrogels were carried out usingAR-G2 rheometer (TA instruments New Castle DE USA)according to a published protocol [14] Briefly after 1minequilibrium time 200120583L PF precursor solution loaded on theinstrument was cross-linked by exposure to long-wave UVlight (365 nm) emitted from OmniCure Series 2000 UV lightsource at an intensity of 5mWcm2 Time-sweep oscillatorytests were done at room temperature at a sinusoidal 2 strainrate and a 6-rad sminus1 angular frequencywithin the linear visco-elastic region as determined previously [15] Temporal changeof storage modulus (1198661015840) was recorded for 3min after initia-tion of cross-linking

24 Swelling Analysis of PF Hydrogels For measuring theswelling ratio PF hydrogels were cast into cylinder moldstransferred to PBS and allowed to equilibrate for 24 h at 4∘CWet weight of the samples was determined before undergo-ing freeze-drying overnight The change in hydrogel weightbetween the swollen (119882

119904) and dried (119882

119889) states was used to

determine the volumetric swelling ratio as follows

Swelling ratio =119882119904

119882119889

(1)

25 DPSC Encapsulation and Differentiation Human DPSCsused in this study were obtained commercially fromAllCellsLLC DPSCs were cultured in Dulbeccorsquos modified eaglersquosmedium (DMEM) (low glucose Biowest France) supple-mented with 10 fetal bovine serum (FBS Biowest) and 1penicillinstreptomycin (PS Life technologies Singapore) Toinitiate cell encapsulation DPSCs at P4-P5 were detached bytrypsinization before being resuspended in PF gel precursorsolution at a density of 1 times 106 cellsmL To initiate cross-linking the cell-gel mixtures were exposed to long-waveUV light for 3min in 15-well 120583-slide or 5min in plasticcylindermoldsDPSCs encapsulated in the PFhydrogelsweredifferentiated to odontoblast-like cells by culturing in odon-togenicosteogenic differentiation medium [16] comprised ofDMEMhigh glucose 10 FBS 1 PS 10minus7Mdexamethasone(Sigma St Louis MO USA) 50120583gmL ascorbic acid 2-phosphate (Sigma) and 10mM 120573-glycerophosphate (Sigma)DPSCs encapsulated in PF hydrogels (cast in both cylindermolds and in 15-well 120583-slides) were differentiated for 3 weekswith medium change every 3 days










3 Results








0

20

40

60

PF-0 PF-05 PF-15 PF-25

Swel

ling

ratio

lowastlowastlowast

lowast









Day 1 Day 7PF

-0PF

-05

PF-1

5PF

-25


0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

Day 1Day 7

PF-0 PF-05 PF-15 PF-25

lowast


PF-0 PF-05

PF-15 PF-25


0

20

40

60

80

100

120

PF-0 PF-05 PF-15 PF-25

Cell

aggr

egat

ion

()

lowastlowastlowast



Col I

1

10

100

7 21

Relat

ive g

ene e

xpre

ssio

n

PF-0PF-05

PF-15PF-25

Days

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

lowastlowastlowast

lowastlowastlowast

(a)

DSPP

PF-0PF-05

PF-15PF-25

1

10

100

1000

10000

100000

7 21

Relat

ive g

ene e

xpre

ssio

n

Days

lowastlowastlowast

(b)

DMP-1

PF-0PF-05

PF-15PF-25

1

10

100

1000

7 21

Relat

ive g

ene e

xpre

ssio

n

Days

lowastlowastlowast

(c)

OC

1

10

100

7 21

Relat

ive g

ene e

xpre

ssio

n

PF-0PF-05

PF-15PF-25

Days

lowastlowastlowast

lowastlowast

lowast

lowast

(d)







0

20

40

60

80

PF-0 PF-05 PF-15 PF-25

lowastlowastlowast

lowastlowast

lowast

(120583g

calc

ium

gel

cons

truc

t)

(a)

0

20

40

60

80

100

PF-0 PF-05 PF-15 PF-25

lowastlowastlowast

lowastlowast

lowastlowast

lowast

(ng

DN

Ag

el co

nstr

uct)

(b)

0

06

12

18

PF-0 PF-05 PF-15 PF-25

lowastlowastlowast

(120583g

calc

ium

ng

DN

A)

(c)

PF-0 PF-05

PF-15 PF-25

Aliz

arin

red


(d)



4 Discussion











5 Conclusions




Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










3 Results








0

20

40

60

PF-0 PF-05 PF-15 PF-25

Swel

ling

ratio

lowastlowastlowast

lowast









Day 1 Day 7PF

-0PF

-05

PF-1

5PF

-25


0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

Day 1Day 7

PF-0 PF-05 PF-15 PF-25

lowast


PF-0 PF-05

PF-15 PF-25


0

20

40

60

80

100

120

PF-0 PF-05 PF-15 PF-25

Cell

aggr

egat

ion

()

lowastlowastlowast



Col I

1

10

100

7 21

Relat

ive g

ene e

xpre

ssio

n

PF-0PF-05

PF-15PF-25

Days

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

lowastlowastlowast

lowastlowastlowast

(a)

DSPP

PF-0PF-05

PF-15PF-25

1

10

100

1000

10000

100000

7 21

Relat

ive g

ene e

xpre

ssio

n

Days

lowastlowastlowast

(b)

DMP-1

PF-0PF-05

PF-15PF-25

1

10

100

1000

7 21

Relat

ive g

ene e

xpre

ssio

n

Days

lowastlowastlowast

(c)

OC

1

10

100

7 21

Relat

ive g

ene e

xpre

ssio

n

PF-0PF-05

PF-15PF-25

Days

lowastlowastlowast

lowastlowast

lowast

lowast

(d)







0

20

40

60

80

PF-0 PF-05 PF-15 PF-25

lowastlowastlowast

lowastlowast

lowast

(120583g

calc

ium

gel

cons

truc

t)

(a)

0

20

40

60

80

100

PF-0 PF-05 PF-15 PF-25

lowastlowastlowast

lowastlowast

lowastlowast

lowast

(ng

DN

Ag

el co

nstr

uct)

(b)

0

06

12

18

PF-0 PF-05 PF-15 PF-25

lowastlowastlowast

(120583g

calc

ium

ng

DN

A)

(c)

PF-0 PF-05

PF-15 PF-25

Aliz

arin

red


(d)



4 Discussion











5 Conclusions




Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





0

20

40

60

PF-0 PF-05 PF-15 PF-25

Swel

ling

ratio

lowastlowastlowast

lowast









Day 1 Day 7PF

-0PF

-05

PF-1

5PF

-25


0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

Day 1Day 7

PF-0 PF-05 PF-15 PF-25

lowast


PF-0 PF-05

PF-15 PF-25


0

20

40

60

80

100

120

PF-0 PF-05 PF-15 PF-25

Cell

aggr

egat

ion

()

lowastlowastlowast



Col I

1

10

100

7 21

Relat

ive g

ene e

xpre

ssio

n

PF-0PF-05

PF-15PF-25

Days

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

lowastlowastlowast

lowastlowastlowast

(a)

DSPP

PF-0PF-05

PF-15PF-25

1

10

100

1000

10000

100000

7 21

Relat

ive g

ene e

xpre

ssio

n

Days

lowastlowastlowast

(b)

DMP-1

PF-0PF-05

PF-15PF-25

1

10

100

1000

7 21

Relat

ive g

ene e

xpre

ssio

n

Days

lowastlowastlowast

(c)

OC

1

10

100

7 21

Relat

ive g

ene e

xpre

ssio

n

PF-0PF-05

PF-15PF-25

Days

lowastlowastlowast

lowastlowast

lowast

lowast

(d)







0

20

40

60

80

PF-0 PF-05 PF-15 PF-25

lowastlowastlowast

lowastlowast

lowast

(120583g

calc

ium

gel

cons

truc

t)

(a)

0

20

40

60

80

100

PF-0 PF-05 PF-15 PF-25

lowastlowastlowast

lowastlowast

lowastlowast

lowast

(ng

DN

Ag

el co

nstr

uct)

(b)

0

06

12

18

PF-0 PF-05 PF-15 PF-25

lowastlowastlowast

(120583g

calc

ium

ng

DN

A)

(c)

PF-0 PF-05

PF-15 PF-25

Aliz

arin

red


(d)



4 Discussion











5 Conclusions




Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Day 1 Day 7PF

-0PF

-05

PF-1

5PF

-25


0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

Day 1Day 7

PF-0 PF-05 PF-15 PF-25

lowast


PF-0 PF-05

PF-15 PF-25


0

20

40

60

80

100

120

PF-0 PF-05 PF-15 PF-25

Cell

aggr

egat

ion

()

lowastlowastlowast



Col I

1

10

100

7 21

Relat

ive g

ene e

xpre

ssio

n

PF-0PF-05

PF-15PF-25

Days

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

lowastlowastlowast

lowastlowastlowast

(a)

DSPP

PF-0PF-05

PF-15PF-25

1

10

100

1000

10000

100000

7 21

Relat

ive g

ene e

xpre

ssio

n

Days

lowastlowastlowast

(b)

DMP-1

PF-0PF-05

PF-15PF-25

1

10

100

1000

7 21

Relat

ive g

ene e

xpre

ssio

n

Days

lowastlowastlowast

(c)

OC

1

10

100

7 21

Relat

ive g

ene e

xpre

ssio

n

PF-0PF-05

PF-15PF-25

Days

lowastlowastlowast

lowastlowast

lowast

lowast

(d)







0

20

40

60

80

PF-0 PF-05 PF-15 PF-25

lowastlowastlowast

lowastlowast

lowast

(120583g

calc

ium

gel

cons

truc

t)

(a)

0

20

40

60

80

100

PF-0 PF-05 PF-15 PF-25

lowastlowastlowast

lowastlowast

lowastlowast

lowast

(ng

DN

Ag

el co

nstr

uct)

(b)

0

06

12

18

PF-0 PF-05 PF-15 PF-25

lowastlowastlowast

(120583g

calc

ium

ng

DN

A)

(c)

PF-0 PF-05

PF-15 PF-25

Aliz

arin

red


(d)



4 Discussion











5 Conclusions




Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Col I

1

10

100

7 21

Relat

ive g

ene e

xpre

ssio

n

PF-0PF-05

PF-15PF-25

Days

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

lowastlowastlowast

lowastlowastlowast

(a)

DSPP

PF-0PF-05

PF-15PF-25

1

10

100

1000

10000

100000

7 21

Relat

ive g

ene e

xpre

ssio

n

Days

lowastlowastlowast

(b)

DMP-1

PF-0PF-05

PF-15PF-25

1

10

100

1000

7 21

Relat

ive g

ene e

xpre

ssio

n

Days

lowastlowastlowast

(c)

OC

1

10

100

7 21

Relat

ive g

ene e

xpre

ssio

n

PF-0PF-05

PF-15PF-25

Days

lowastlowastlowast

lowastlowast

lowast

lowast

(d)







0

20

40

60

80

PF-0 PF-05 PF-15 PF-25

lowastlowastlowast

lowastlowast

lowast

(120583g

calc

ium

gel

cons

truc

t)

(a)

0

20

40

60

80

100

PF-0 PF-05 PF-15 PF-25

lowastlowastlowast

lowastlowast

lowastlowast

lowast

(ng

DN

Ag

el co

nstr

uct)

(b)

0

06

12

18

PF-0 PF-05 PF-15 PF-25

lowastlowastlowast

(120583g

calc

ium

ng

DN

A)

(c)

PF-0 PF-05

PF-15 PF-25

Aliz

arin

red


(d)



4 Discussion











5 Conclusions




Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

20

40

60

80

PF-0 PF-05 PF-15 PF-25

lowastlowastlowast

lowastlowast

lowast

(120583g

calc

ium

gel

cons

truc

t)

(a)

0

20

40

60

80

100

PF-0 PF-05 PF-15 PF-25

lowastlowastlowast

lowastlowast

lowastlowast

lowast

(ng

DN

Ag

el co

nstr

uct)

(b)

0

06

12

18

PF-0 PF-05 PF-15 PF-25

lowastlowastlowast

(120583g

calc

ium

ng

DN

A)

(c)

PF-0 PF-05

PF-15 PF-25

Aliz

arin

red


(d)



4 Discussion











5 Conclusions




Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








5 Conclusions




Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article modulation of dental pulp stem...

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