Basic Research—Biology

Mechanical Changes in Human Dental Pulp Stem Cellsduring Early Odontogenic DifferentiationTaneka D. Jones, BS, MS,* Hamed Naimipour, BS, MS,* Shan Sun, PhD,* Michael Cho, PhD,*and Satish B. Alapati, BDS, MS, PhD†

Abstract

Introduction: Cell adhesion and migration in bioactivescaffolds require actin cytoskeleton remodeling andfocal adhesion formation. Additionally, human dentalpulp stem cells (hDPSCs) undergo several changes intheir mechanical properties during odontogenic differ-entiation. The effect of factors essential for odontogen-esis on actin stress fiber elasticity and focal adhesionformation is not known. Methods: Live hDPSCscultured in odontogenic media were imaged for cyto-skeleton changes using an atomic force microscope.The Young’s modulus (kPa) of the cytoskeleton was re-corded as a function of culture medium for 10 days.Focal adhesion formation was assessed using immuno-fluorescence. Cultured hDPSCs were incubated with amonoclonal vinculin antibody, and filamentous actinswere visualized using 0.5 mmol/L phalloidin. Results:Cytoskeletal elasticity significantly increased inresponse to odontogenic media. Both the number andphysical size of focal adhesions in hDPSCs alsoincreased. Up-regulation of vinculin expression wasevident. The increase in the formation of focal adhesionswas consistent with actin remodeling to stress fibers.Conclusions: Our findings suggest that hDPSCs firmlyattach to the glass substrate in response to odontogenicmedia. Successful regeneration of pulp-dentin tissueusing biomimetic scaffolds will likely require cell-extracellular matrix interactions influenced by biochem-ical induction factors. (J Endod 2014;-:1–6)

Key WordsActin reorganization, adhesion, attachment, humandental pulp stem cells, regeneration

From the Departments of *Bioengineering and†Endodontics, University of Illinois at Chicago, Chicago, Illinois.

Address requests for reprints to Dr Satish B. Alapati,Department of Endodontics, University of Illinois at Chicago,College of Dentistry, 801 South Paulina (M/C 642), Room536B, Chicago, IL 60612. E-mail address: salapati@uic.edu0099-2399/$ - see front matter

Copyright ª 2014 American Association of Endodontists.http://dx.doi.org/10.1016/j.joen.2014.07.030

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Promising options for the formation of reparative dentin include direct pulp capping(1) and human dental pulp stem cell (hDPSC) transplantation. Both alternatives to

root canal therapy involve the interaction of a cell source with an extracellular bioactivematerial (2). Extracellular matrix (ECM) scaffolds and inductive chemical factors inculture medium may affect cell adhesion, migration, proliferation, and differentiation.Importantly, cellular actin cytoskeleton reorganization and focal adhesion formationhave been shown to impact differentiation in human mesenchymal stem cells (2, 3).

Adherent cells, including hDPSCs, must attach to a substrate before differentiatinginto terminal odontoblastlike cells (3), and inductive cues in culture medium couldcause mechanical changes in hDPSCs affecting cell fate. Focal adhesion protein com-plexes are essential for cell attachment and migration (3, 4). Vinculin, a structuraladaptor protein found in focal adhesion complexes, anchors the actin cytoskeletonto signaling proteins that communicate with the ECM (5). The actin cytoskeleton isan important regulator of cell body elasticity and plasmamembrane signal transductionin response to external stimuli such as soluble factors and substrate elasticity (6–8).Stress fiber formation resulting in cytoskeleton reorganization and subsequent tissueremodeling affects stem cell viability, self-renewal, and differentiation (9, 10).

Recent studies have shown that pulp capping materials, such as mineral trioxideaggregate and BioAggregate (Innovative Bioceramix, Vancouver, BC, Canada), enhancethe association of focal adhesion and actin stress fibers in vitro (2). Actin remodelinghas been shown to mediate decreased cell elasticity in human mesenchymal stem cellssubjected to osteodifferentiation factors (10–13). However, the role of odontogenicdifferentiation factors in altering the cytoskeletal organization in hDPSCs remainsunknown. To address this, we examined changes in vinculin expression and cellstiffness (Young’s modulus) upon exposure to odontogenic biochemical factorsin vitro.

Although there is no standardized induction cocktail for the odontoblastlike dif-ferentiation of hDPSCs, the protocol reported by Gronthos et al (14) has been widelyused (15). Using this induction protocol, we report key quantitative data for dental ma-terial and tissue engineering applications based on novel mechanical characterization.Our aimwas to validate the hypothesis that soluble factors in odontogenic differentiationmedium affect hDPSC cytoskeleton elasticity and focal adhesion formation. Thisresearch will inform future experiments designed to optimize the fabrication of biomi-metic scaffolds for hDPSC differentiation requiring initial soluble factors in vitro.

Materials and MethodsCell Culture

hDPSCs (donors aged 18–25 years) were kindly gifted from Dr. Songato Shiat the University of Southern California, Los Angeles, CA. Cells were cultured in alphaminimum essential medium supplemented with 20% fetal bovine serum, 2 mmol/LL-glutamine, 100 mmol/L L-ascorbic acid 2-phosphate, 100 U/mL penicillin, and100 mg/mL streptomycin (14). Passage 4 cells were incubated at 37�C in a 5%CO2, and the culture medium was exchanged every other day.

Odontogeniclike InductionCells between passages 4 and 5 were incubated with odontogenic induction media

containing alpha minimum essential medium supplemented with 20% fetal bovine

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serum, 2 mmol/L L-glutamine, 100 mmol/L L-ascorbic acid 2-phosphate, 100 U/mL penicillin and 100 mg/mL streptomycin,1.8 mmol/L monopotassium phosphate, KH2PO4, and 10 nmol/L dexa-methasone (14). Samples were subcultured onto 22 � 22 mm glasscoverslips at a density of 3 � 103 cells/cm2. Samples for cell elasticityexperiments were cultured on days 1, 3, 7, and 10. Samples for focaladhesion experiments were sampled over 5 days.

Cell Elasticity MeasurementsThe cell elasticity of live hDPSCs was measured with an atomic

force microscope (Novascan Technologies, Ames, IA) using the micro-indentation technique. Briefly, a silicon nitride cantilever probe (Veeco,Santa Barbara, CA) 100 mm in length was positioned over the cyto-plasmic region of live hDPSCs in phosphate buffered saline avoidingthe perinuclear region. Each cell was indented with the atomic forcemicroscopic probe at 3 different locations over a 15 � 15 mm area.Cells grown on days 1, 3, 7, and 10 were measured. A force curvewas obtained by measuring the deflection of the cantilever correspond-ing to the applied force. Force-distance curves were analyzed using theHertz model (10) as follows:

F ¼ 4

3

E

ð1� y2Þd32OR

where F is the loading force, is the Young’s elastic modulus, n is thecellular Poisson’s ratio, d is the cell indentation depth, and R is theradius of the spherical indenter (5 mm). The average Young’s modulusfor the hDPSCs cultured in odontogenic and control culture medium at4 time points was calculated and analyzed using 2-way analysis of vari-ance followed by the Friedman and Kruskal-Wallis tests.

Immunofluorescence and Fluorescent MicroscopyTo observe cytoskeletal actin microfilaments, hDPSCs were fixed

with 4% formalin and permeabilized in cold (�20�C) acetone for 3 mi-nutes. A 5% bovine serum albumin (BSA) solution was applied to thesamples to block nonspecific binding sites for 30 minutes at room tem-perature. Actin microfilaments were stained with rhodamine phalloidin(5 mmol/L) (Molecular Probes, Eugene, OR) for 30 minutes at roomtemperature. Samples were imaged with a fluorescent microscope ondays 0, 1, 7, and 10. To determine the effect of actin filament disruptionon cell elasticity, the cytoskeleton was disrupted with cytochalasin D(5 mm) (Sigma-Aldrich, St Louis, MO) after incubation for 30 minutesat 37�C (10). Samples for focal adhesion observations were incubatedwith mouse antihuman primary vinculin antibody overnight at 4�C andfurther treated with fluorescein isothiocyanate (FITC)-conjugated(green fluorescence) goat antimouse secondary antibody for 1 hourat 37�C (Millipore, Billerica, MD). F-actin was stained simultaneouslywith 0.5 mmol/L tetramethylrhodamine (TRITC) conjugated (red fluo-rescence) phalloidin (Millipore). Nuclei staining (blue fluorescence)was visualized using 0.5 mmol/L 40,6-diamidino-2-phenylindole for3 minutes at room temperature. Staining was conducted on days 1, 3,and 5 in control and odontogenic induction medium in triplicate. Sam-ples treated for immunocytochemistry were visualized with an E-800Eclipse Nikon fluorescent microscope (Nikon, Melville, NY) with a60� objective lens and a 16-bit charge-coupled device camera (Pho-tometrics, Tucscon, AZ). Images were pseudocolored with MetaMorphsoftware (Molecular Devices, Downingtown, PA).

Statistical AnalysisAll values of statistical analysis are expressed as average

values � standard deviation. Statistical analysis of hDPSC cytoskeleton

2 Jones et al.

elasticity and focal adhesion formation was performed using 2-wayanalysis of variance followed by the Friedman and Kruskal-Wallis tests.Actin disruption data were analyzed using the Student t test. P values#.05 were considered significant.

ResultsEffect of Odontogenic Induction Mediumon hDPSC Cytoskeleton Elasticity

To address whether odontogenic differentiation medium affectscytoskeletal changes, we examined hDPSC actin stress fiber formationusing atomic force microscopy. Exposure time periods were selectedto allow sufficient time for lineage commitment. As shown inFigure 1A, cells exposed to odontogenic medium for 7 and 10 daysshowed a marked increase in thickness when compared with 0 and 1day. Additionally, the Young’s modulus, as measured using the atomicforcemicroscopicmicroindentation technique, exhibited an increase inkPa (4.11) in cells exposed to odontogenic medium on day 10. Incontrast, cells exposed to odontogenic medium on days 1 and 3 showeda significantly less modulus (2.25 kPa) (Fig. 1B). The increase inYoung’s modulus over time was significantly greater (P # .05) inhDPSCs cultured in odontogenic medium.

Influence of Cytoskeleton Disruptionon the Average Young's Modulus

In continuation with our findings in Figure 1B, we intended to testwhether the disruption of the cytoskeleton hinders the cell stiffness inhDPSCs. Interestingly, as observed in Figure 2A, cells exposed to cyto-chalasin D showed almost no stress fibers. Furthermore, cells treatedwith cytochalasin D showed a significant reduction (P # .05) inYoung’s modulus (kPa) when compared with cells treated with differ-entiation media alone (Fig. 2B).

Odontogenic Differentiation IncreasesFocal Adhesion Proteins in hDPSCs

In our next set of experiments, we tested whether the differentia-tion of hDPSCs toward an odontogenic lineage affects focal adhesionpresence and localization. We performed the experiment at 3 differenttime points: 1, 3, and 5 days. In our experiments, we stained for vincu-lin. Interestingly, our findings suggest an increase in the intensity of fluo-rescence, representing the levels of vinculin in hDPSCs (Fig. 3A). Anincrease in the number of vinculin molecules was observed in cellstreated with odontogenic medium. The average number of focal adhe-sions per cell in the microscopic field of view in experimental samplessignificantly increased (P# .05) over time compared with the controlsamples (Fig. 3B).

DiscussionRecent research reports pulp capping and cell-based therapies as

possible methods to regenerate compromised pulp tissue. Pulp cappinghas been shown to maintain pulp vitality and enhance dentin repairmechanisms in damaged pulp tissue (1). Conversely, stem cells isolatedfrom human dental pulp have been combined with cell delivery scaf-folds for pulp regeneration applications (15). Odontoblastlike differen-tiation using hDPSCs and bioengineered ECM scaffolds may addressshortcomings associated with pulp capping materials such as limitedreparative dentin formation (16) and, thus, the understanding thatthe effect of odontongenic media on cell-ECM interactions in vitro isnecessary for in vivo comparison.

Cell adhesion, migration, and differentiation are 3 types of cell-ECM interactions influenced by actin cytoskeletal reorganization and

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Figure 1. (A) Actin filament reorganization of hDPSCs exposed to odontogentic medium at 4 time points as determined by phalloidin immunofluorescence stain-ing. Scale bar = 30 mm. Images represent samples from 3 independent experiments. (B) The effect of odontogenic medium on Young’s modulus of hDPSCs asquantified by using atomic force microscopy.

Basic Research—Biology

focal adhesion formation (10, 17). The contribution of solubledifferentiation biochemical factors, such as dexamethasone, to thesemechanical responses is unclear. Cell adhesion is required for cellattachment to a scaffold for adherent cell types (2, 18), and our studyreports the changes in the hDPSC cytoskeletal elasticity and vinculinprotein expression of hDPSCs during early odontogenic differentiation.

In this study, we define early odontogenic differentiation as sam-ples exposed to induction medium for less than 10 days. hDPSCs

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cultured on glass coverslips showed an increase in cytoskeletal elastic-ity. The Young’s modulus increased in live hDPSCs induced with odon-togenic medium from days 1–7, with an average increase between 3 and4 kPa.

The actin stress fibers also thickened as time progressed, re-placing initial thinner actin meshworks. This may be caused by thedexamethasone in the induction medium penetrating the cell mem-brane and initiating an odontoblastlike lineage (3). The decrease

Mechanical Changes in hDPSCs 3

Figure 2. (A) The effect of cytochalasin D treatment on the cytoskeleton of hDPSCs. Scale bar = 30 mm. Images represent samples from 3 independent exper-iments. (B) The effect of ctyochalasin D treatment on the Young’s modulus of hDPSCs.

Basic Research—Biology

in the modulus on day 10 may have been caused by changes ingene expression as previously reported by Titushkin and Cho(10). Sample treatment with cytochalasin D significantly decreasedthe average elastic modulus of hDPSCs, most likely because of de-polymerized stress fibers. These data show that actin filamentsregulate cell stiffness.

Interestingly, human mesenchymal stem cells induced toward anosteogenic lineage with 10 nmol/L dexamethasone between days 1 and7 resulted in thick actin stress fibers replaced with thinner actin fila-ments with a decrease in Young’s modulus on day 10 (10). Additionally,the concentration of dexamethasone used to induce hDPSCs toward anodontoblastlike lineage was 10 nmol/L as previously reported by Gron-thos et al (14). The results suggest that, despite similarities in geneexpression, profiles between the 2 cell types (19) and the mechanicalproperties may be modulated differently. This hypothesis can be testedin future studies by comparing overlapping odontogenic and osteogenicgene markers at different time points with multiple hDPSC cell lines anddonors.

Focal adhesion formation is also required for cell adhesionand migration (17). Focal adhesions are flat, elongated structuresranging from 2–5 mm in size (20-24). Our findings suggest thathDPSCs become strongly attached to the substrate in responseto the odontogenic factors. We used immunofluorescent stainingto map the presence of vinculin, a 117-kd protein, at the hDPSCperiphery. The mean number of focal adhesions per cell increasedlinearly with time for the samples exposed to odontogenic mediumbut did not change in the control cells between days 3 and 5. This

4 Jones et al.

was potentially caused by strong adhesions formed on the rigidglass restricting cell migration. Conversely, soluble signals pro-moting migration in the induction media or changes in intercel-lular contact may have contributed to the increased averagenumber of focal adhesions in hDPSCs cultured in the odontogenicmedium (25-27).

One method to better characterize this interaction would be tosuppress focal adhesion kinase to determine if focal adhesion formationis inhibited at extended time points. Additionally, identifying the key in-tegrins expressed by hDPSCs as a function of substrate stiffness wouldprovide insight into the molecular interactions that regulate mechanicalevents. Integrin expression is necessary for the adherence of cells tosynthetic scaffolds (22). A recent study examining stress fiber assemblyand dental pulp cell adhesion formation to different pulp capping agentsshowed preferential focal adhesion to BioAggegrate, highlighting theclinical significance of this work (2).

In summary, the development of optimal scaffolds for dentalpulp tissue engineering will likely require synergy between inductivefactors in culture medium, scaffold material, and cell sources. Thecurrent study shows that the elasticity of the hDPSC cytoskeleton in-creases over time and focal adhesion formation is enhanced by odon-togenic medium. An important unknown factor is how hDPSCs mayrespond to physical scaffold cues in the absence of induction mediumin vivo. The next step in the use of data quantifying mechanicalchanges in differentiating hDPSCs would be to seed hDPSCs on scaf-folds of varying matrix elasticity because this has also been shown todirect stem cell lineage (3).

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Figure 3. (A) Focal adhesion formation exposed to odontogenic induction medium as determined by immunofluorescence staining. hDPSC were treated withodontogenic medium for 5 days. Vinculin (green) was colocalized with the actin cytoskeleton and stained with phalloidin (red). Scale bar = 35 mm. Images repre-sent samples from 3 independent experiments. (B) The average number of focal adhesions per cell as a function of culture medium over time.

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AcknowledgmentsThe authors thank Dr Amelia Zellander for her technical assis-

tance with sample preparation.Supported by a NIH/NIDCR grant (grant no. DE019514-SBA).The authors deny any conflicts of interest related to this study.

References1. Cho SY, Seo DG, Lee SJ, et al. Prognostic factors for clinical outcomes according to

time after direct pulp capping. J Endod 2013;39:327–31.2. Zhu L, Yang J, Zhang J, et al. A comparative study of BioAggregate and ProRoot MTA

on adhesion, migration, and attachment of human dental pulp stem cells. J Endod2014;40:1118–23.

3. Engler A, Sen S, Sweeney H, et al. Matrix elasticity directs stem cell lineage specifi-cation. Cell 2006;126:677–89.

4. Yamada K, Geiger B. Molecular interactions in cell adhesion complexes. Curr OpinCell Biol 1997;9:76–85.

5. Matsudaira P. Modular organization of actin crosslinking proteins. Trends BiochemSci 1991;16:87–92.

6. Matsudaira P. Actin crosslinking proteins at the leading edge. Semin Cell Biol 1994;5:165–74.

7. Burridge K, Nuckolls G, Otey C, et al. Actin-membrane interaction in focal adhe-sions. Cell Differ Dev 1990;32:337–42.

8. Turner C, Burridge K. Transmembrane molecular assemblies in extra-cellular inter-actions. Curr Opin Cell Biol 1991;3:849–53.

9. Burridge K, Chrzanowska-Wodnicka M. Focal adhesions, contractility, andsignaling. Annu Rev Cell Dev Biol 1996;12:463–518.

10. Titushkin I, Cho M. Modulation of cellular mechanics during osteogenic dif-ferentiation of human mesenchymal stem cells. Biophys J 2007;93:3693–702.

11. Ghosh K, Pan Z, Guan E, et al. Cell adaptation to a physiologically relevant ECMmimic with different visoelastic properties. Biomaterials 2007;28:671–9.

6 Jones et al.

12. Solon J, Levental I, Sengupta P, et al. Fibroblast adaptation and stiffness matching tosoft elastic substrates. Biophys J 2007;93:4453–61.

13. Yu X, Bellamkonda R. Dorsal root ganglia neurite extension is inhibited by mechan-ical and chrondroitin sulfate-rich interfaces. J Neurosci Res 2001;66:303–10.

14. Gronthos S, Mankani M, Brahim J, et al. Postnatal human dental pulp stem cells(DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A 2000;97:13625–30.

15. Mao J, Kim S, Zhou J, et al. Regenerative endodontics: barriers and strategies forclinical translation. Dent Clin North Am 2012;56:639–49.

16. Iohara K, Nakashima M, Ito M, et al. Dentin regeneration by dental pulp stem celltherapy with recombinant human bone morphogenetic protein 2. J Dent Res 2004;83:590–5.

17. Pelham R, Wang Y. Cell locomotion and focal adhesions are regulated by substrateflexibility. Proc Natl Acad Sci U S A 1997;94:13661–5.

18. Galler K, D’Souza R, Hartgerink J, et al. Scaffolds for dental pulp tissue engineering.Adv Dent Res 2011;23:333–9.

19. Shi S, Robey P, Gronthos S. Comparison of human dental pulp and bone marrowstromal cells by cDNA microarray analysis. Bone 2001;29:532–9.

20. Geiger B, Spatz J, Bershadsky A. Environmental sensing through focal adhesions. NatRev Mol Cell Biol 2009;10:21–33.

21. Geiger B, Bershadsky A, Pankov R, et al. Transmembrane crosstalk between theextracellular matrix and the cytoskeleton. Nat Rev Mol Cell Biol 2001;2:793–805.

22. Kaverina I, Krylyshkina O, Small J. Regulation of substrate adhesion dynamics duringcell motility. Int J Biochem Cell Biol 2002;34:746–61.

23. Pollard T, Borisy G. Cellular motility driven by assembly and disassembly of actinfilaments. Cell 2003;112:453–65.

24. Zaidel-Bar R, Ballestrem C, Kam Z, et al. Early molecular events in the assembly ofmatrix adhesions at the leading edge of migrating cells. J Cell Sci 2003;116:4605–13.

25. Lutolf M, Hubbell J. Synthetic biomaterials as instructive extracellular microenvi-ronments for morphogenesis in tissue engineering. Nat Biotechnol 2005;23:47–55.

26. Ridley A, Schwartz M, Burridge K, et al. Cell migration: integrating signals from frontto back. Science 2003;302:1704–9.

27. O’Neill G. The coordination between actin filaments and adhesion in mesencyhmalmigration. Cell Adh Migr 2009;3:355–7.

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