dental pulp stem cell migration

Basic Research—Biology

Dental Pulp Stem Cell MigrationCameron Howard, DMD,* Peter E. Murray, PhD,† and Kenneth N. Namerow, DDS†

Abstract
Introduction: The purpose of this in vitro study wasto investigate the migration of dental pulp stem cells(DPSCs) in response to chemotactants and extracellularmatrix proteins (EMPs). This DPSC signaling informationis needed to help understand tooth regeneration afterinjury and to develop some future regenerativeendodontic therapies. Methods: DPSCs were releasedby trypsinization and plated on transwell filters. The che-motactants were recombinant sphingosine-1-phosphate(S1P), fibroblast growth factor (FGF), epidermal growthfactor (EGF), or transforming growth factor beta-1 (TGF-b1), and the EMPs were collagen-1, collagen-IV, lami-nin, and fibronectin. Data were analyzed by using anal-ysis of variance (ANOVA) statistical tests for cellmigration. Results: S1P induced more vigorous DPSCmigration in comparison with the other TGF- b1, FGF,or EFG chemotactants (ANOVA, P < .05). Laminininduced more vigorous DPSC migration in comparisonwith the other EMPs (ANOVA, P < .05). Conclusions:The EMPs, particularly laminin, and chemotactants,particularly S1P and TGF-b1, were found to be importantpromoters of DPSC migration. The interplay between theEMPs, blood lipid, serum, and chemotactants suggeststhat the migration of DPSC is highly regulated. Specificchemotactants and EMPs might mediate the processof pulp-dentin regeneration after tooth injury, andthey could be used as part of regenerative endodontictherapy. (J Endod 2010;36:1963–1966)
Key WordsCell signaling, chemotactants, chemotaxis, extracellularmatrix, growth factors

From *Private Practice, Tampa, Florida; and †College ofDental Medicine, Nova Southeastern University, Fort Lauder-dale, Florida.

Address requests for reprints to Dr Peter E. Murray, Depart-ment of Endodontics, College of Dental Medicine, Nova South-eastern University, 3200 S University Dr, Fort Lauderdale, FL33328-2018. E-mail address: [email protected]/$ - see front matter

Copyright ª 2010 American Association of Endodontists.doi:10.1016/j.joen.2010.08.046

JOE — Volume 36, Number 12, December 2010

Cell migration is necessary for homeostatic tissue maintenance and the regenerationof injured organs and tissues (1). Cells must migrate to sites where they are

required to function, otherwise the regeneration and development of functional organsand tissues would be impossible (2). Conversely, deregulated cell migration can resultin the development of a number of pathologies, including tumor metastasis and angio-genesis, chronic inflammation, and various immune response dysfunctions (3). Aspecial population of cells called dental pulp stem cells (DPSCs) (4), odontoblast-like cells (5), or odontoblastoid cells (6) from adult teeth or stem cells from humanexfoliated deciduous teeth (SHED) (7) are known to respond to tooth injury by prolif-erating, migrating, and differentiating to replace lost odontoblasts, leading to thesynthesis and secretion of tertiary dentin (8) often called a dentin bridge (9). Thespecific chemotactants that cause the DPSCs to respond to tooth injury have notbeen identified.

The dentin matrix is a reservoir of chemotactants secreted by odontoblasts andpulp fibroblasts. The potential chemotactants include transforming growth factor b1(TGF-b1), fibroblast growth factors (FGFs), and epidermal growth factors (EGFs).These chemotactants are hypothesized to provide the signals involved in the prolifera-tion, differentiation, and recruitment of pulp cells to the site of tooth injury to initiatetissue regeneration (10, 11). Alternatively, other potential chemotactants such as thenaturally occurring bioactive blood-borne sphingolipid mediator, sphingosine-1-phosphate (S1P), have important functions in inflammation and wound healing suchas differentiation, proliferation, survival, and angiogenesis (12) and might direct cellmigration in response to tooth injury. However, the relative potency of these chemotac-tants to promote the migration of DPSCs is not known.

Cells are known to sense and respond to their local environment by using integrinreceptor-mediated signaling pathways, in particular to sense the biochemical propertiesof the extracellular matrix proteins (EMPs) (13). The complex interactions of cells withEMPs play crucial roles in mediating and regulating cell adhesion, migration, morpho-genesis, tissue homeostasis, tumorigenesis, and wound healing (14). The main EMPsare collagen, laminin, and fibronectin (15). Laminin is essential for odontoblast differ-entiation (16). Fibronectin promotes ameloblast growth and differentiation (17).Collagen is the most ubiquitous EMP; it has an affinity for binding and immobilizinggrowth factors to regulate cell proliferation and differentiation (18). Collagen and lam-inin are known to promote themigration of osteoblast cells (19), but the effects of EMPson the migration of DPSCs have not yet been established.

The purpose of this in vitro study was to investigate the migration of DPSCs inresponse to chemotactants and EMPs to collect the basic signaling information neededto help develop some future regenerative endodontic therapies.

Materials and MethodsSources of Cell Lines

A DPSC line (SHED) was provided under a material transfer agreement with theNational Institute of Dental and Craniofacial Research (7). Human dermal microvas-cular endothelial cells (endothelial cells) were obtained from Clonetics/Biowhittaker(Walkersville, MD). Human aortic smooth muscle cells (smooth muscle cells, cloneACBR1716) were obtained from Cell Systems Corp (Kirkland, WA).

Cell CultureThe DPSCs were cultured in Dulbecco modified Eagle medium (DMEM) (Clo-

netics/Biowhittaker, Emerainville, France) supplemented with 10% fetal bovine serum

Pulp Cell Migration 1963

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http://dx.doi.org/10.1016/j.joen.2010.08.046

(FBS) (Hyclone, Logan, UT) and 1% gentamicin and amphotericin anti-biotic. The endothelial cells were cultured in EGM-2 MV media (Clo-netics, San Diego, CA) supplemented with microvascular endothelialcell growth medium–2, which contains recombinant growth factors,ascorbate, hydrocortisone, insulin-like growth factor, heparin, 15%FBS, and 1% antibiotics (gentamicin and amphotericin). The smoothmuscle cells were cultured in DMEM supplemented with 10% FBSand 1% antibiotics.
The cells were grown in T-75 tissue culture treated plastic flasks(BD Falcon; BD Biosciences, San Jose, CA) maintained in an atmo-sphere of 5% CO2 and grown to 95% contact-inhibited monolayerswith typical cobblestone morphology (7). Before analysis, the cellswere starved in the serum-free and growth factor–free culture mediafor 3 hours. The cells were detached by incubation with 0.5%trypsin/0.2% ethylenediaminetetraacetic acid and pelleted by centrifu-gation; the pellets were washed gently and resuspended in serum-freeand growth factor–free culture media at a concentration of 3 � 106

cells/mL.

Figure 1. Mean DPSC migration in response to EMPs. In the control group(none) no protein was added. Error bars represent the standard deviationof the mean.

Migration of CellsThe cells (3 � 106 cells/mL) were aliquoted within 100 mL of

serum-free and growth factor–free culture media into the uppercompartment of a 6-mm-diameter migration chamber (Costar Trans-well; Corning Inc, Corning, NY) that was pre-warmed to 37�C. Theupper migration chamber had a base composed of a polycarbonatefilter containing 8-mm pores. The filter was precoated with laminin-1, fibronectin, collagen-1, or collagen-IV (Sigma, St Louis, MO) ata concentration of 10mg/100 mm2 surface area. The cells were allowedto adhere to the filters for 2 hours at 37�C in a moist environment and5% CO2 atmosphere. After cell adherence, the upper migration cham-bers were placed into lower chambers containing growth factors in theserum-free and growth factor–free culture media (20).

The optimal concentrations of chemotactants to promote DPSCmigration were determined by using dose-response curves between1 ng/mL to 500 mm/mL. The concentrations of target growth factorsin the lower migration chambers were S1P (500 mm) (Avanti PolarLipids, Alabaster, AL), TGF-b1 (10 ng/mL), FGFs (10 ng/mL), andEGFs (10 ng/mL) (all supplied by Sigma Chemical Co). FBS was addedto the culture media in the lower migration chambers at a concentra-tion of 10% v/v. Those solutions were prepared fresh immediatelybefore experimentation to prevent gradient formation. In the controlgroup (none) no growth factor was added. The cells were incubatedfor 4 hours.

Figure 2. Mean DPSC migration in response to growth factors of fetal calfserum (serum), EGF, FGF, TGF-b1, and S1P by using a transwell chambermigration assay. In the control group (none) no growth factor was added.Error bars represent the standard deviation of the mean.

Numbers of Migrating CellsAfter migration, the upper chamber containing the filter was

removed, and the cells were fixed with 10% neutral-buffered formalin(Fisher Scientific, Nepean, ON, Canada) for 10 minutes. The non-migrating cells attached to the upper side of the filter were carefullyremoved with a cotton swab, and the migrating cells attached to thelower side of the filter were stained with hematoxylin for 60 seconds.The numbers of migrating cells were counted per microscope field at�200 magnification by using checkerboard analysis (20) with a blindcoded specimen technique for each of the different treatment protocols.Each of the individual treatment protocols for each of the growthfactors, FBS, EMPs, and negative control (no growth factor) were repli-cated 10 times. The numbers of attached cells were counted andanalyzed by using one-way analysis of variance (ANOVA) statistical tests.The raw data were evaluated by using STATview software (SAS Inc, Cary,NC) at a confidence level of 95%.

1964 Howard et al.

ResultsThe polycarbonate migration filters were used uncoated and also

coated with EMPs. The coating of filters with laminin at 10 mg/100 mm2

surface area provided the optimal DPSC migration in response to 500mmol/L of S1P (Fig. 1) compared with the other chemotactants (AN-OVA, P < .05). In comparison to the uncoated filters, the EMPsincreased DPSC migration by the following amounts: collagen-IV(7.9%), collagen-1 (61.0%), fibronection (93.9%), and laminin(313.1%) (Fig. 1).

The optimal concentrations of chemotactants to promote DPSCmigration were determined by using dose-response curves. The optimalconcentrations of chemotactants on laminin-coated filters to promotemigration were 500 mmol/L for S1P and 10 ng/mL for TGF-b1, FGFs,and EGFs. There were differences in the numbers of migrating DPSCscounted on the filters dependent on the target chemotactants (ANOVA,P < .05), shown in Fig. 2. The greatest numbers of migrating DPSCs


Figure 3. Comparison of cell migration responses to concentrations of S1P.Scatter diagram with lines of best fit for each cell type shows the mean migra-tion of DPSCs, endothelial cells, and smooth muscle cells in response toconcentrations of S1P.


were counted on the filters in response to S1P in comparison with theother chemotactants: TGF-b1, FGFs, or EFGs (ANOVA, P < .05). Incomparison to the absence of chemotactant (negative control treat-ment), the chemotactants increased DPSC migration by the followingamounts: serum (–26.3%), EGFs (19.9%), FGFs (70.4%), TGF-b1(131.2%), and S1P (258.6%) (Fig. 2).

The migration of DPSCs, endothelial cells, and smooth musclecells was concentration-dependent to S1P on laminin-coated filters,although the migration response of DPSCs was different to both theother cell types (ANOVA, P < .05), as shown in Fig. 3. Relatively highconcentrations of S1P (500mmol/L) promoted the migration of DPSCs,whereas lower concentrations (100, 10, and 5mmol/L) of S1P reducedthe numbers of migrating DPSCs. The concentration of S1P had theopposite effects on themigration of endothelial cells and smoothmusclecells (Fig. 3).

DiscussionThis is the first study to demonstrate that the directional migra-

tion of DPSCs is mediated by both chemotactants and EMPs. Themigration of DPSCs in response to chemotactants and EMPs explainstheir ability to detect tooth injury and to perform regeneration (10,11). Previous research has already identified growth factors, suchas TGF-b1 sequestered in the dentin matrix (21), which might func-tion as chemotactic gradients for DPSC migration after injury (10).However, the relative potency of some common chemotactants onDPSC migration was not previously known. The present study demon-strated that TGF-b1 can stimulate DPSC migration, but it was a lesspotent chemotactant than the blood-borne lipid S1P (12). The otherchemotactants; EGFs and FGFs, also have some potential to stimulateDPSC migration, but their potency was relatively weak in comparisonto TGF-b1 or S1P.

The authors included bovine serum in this study because they ex-pected the cocktail of growth factors it contained to promote DPSCmigration, but instead it had a negative effect on DPSC migration.This might suggest that the presence of circulating serum in the bloodsupply of an in situ healthy tooth holds themigration and differentiationof DPSCs in check, thereby preventing their activation and migration,whereas the presence of solubilized TGF-b1 from the damaged dentin


matrix and S1P from the increased vascular supply in traumatized teethcan stimulate DPSC migration.

The injury of odontoblasts and endothelial cells has been demon-strated to recruit DPSCs to the site of injury (22, 23). This providessome evidence to suggest that DPSCs can sense and respond tochanges in local environment conditions. Most cell types are able todetect the biochemical properties of the EMPs (13). The complex inter-actions of cells with the EMPs can play crucial roles in mediating cellmigration (14). The present study is the first to identify laminin as beingthe most effective EMP to promote the migration of DPSCs. Laminin wasalready identified as being essential for odontoblast differentiation(16). The most ubiquitous EMP component is collagen; in the presentstudy collagen-1 and collagen-IV were not very effective promoters ofDPSC migration. A previous study found that both laminin and collagenpromote the migration of osteoblast cells (19). Increased lamininimmunoreactivity has been reported in healing replanted teeth (24).

In conclusion, the EMPs, particularly laminin, and chemotactants,particularly S1P and TGF-b1, were found to be important promoters ofDPSC migration. The interplay between the EMPs, blood lipid, serum,and chemotactants suggests that the migration of DPSCs is highly regu-lated. Specific chemotactants and EMPs might mediate the process ofpulp-dentin regeneration after tooth injury, and they could be usedas part of regenerative endodontic therapy.

AcknowledgmentsThis study was supported by the AAE Foundation.The authors deny any conflicts of interest related to this study.

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