dental pulp tissue engineering with stem cells from exfoliated deciduous teeth

Dental Pulp Tissue Engineering with Stem Cells fromExfoliated Deciduous TeethMabel M. Cordeiro, DDS, MS, PhD,* Zhihong Dong, MD, PhD,*Tomoatsu Kaneko, DDS, PhD,*† Zhaocheng Zhang, MD, PhD,*Marta Miyazawa, DDS, MS, PhD,* Songtao Shi, DDS, PhD,‡ Anthony J. Smith, BSc, DDS, PhD,§

and Jacques E. Nör, DDS, MS, PhD*�

AbstractStem cells from human exfoliated deciduous teeth(SHED) have been isolated and characterized as multi-potent cells. However, it is not known whether SHEDcan generate a dental pulp-like tissue in vivo. Thepurpose of this study was to evaluate morphologiccharacteristics of the tissue formed when SHED seededin biodegradable scaffolds prepared within humantooth slices are transplanted into immunodeficientmice. We observed that the resulting tissue presentedarchitecture and cellularity that closely resemble thoseof a physiologic dental pulp. Ultrastructural analysiswith transmission electron microscopy and immunohis-tochemistry for dentin sialoprotein suggested thatSHED differentiated into odontoblast-like cells in vivo.Notably, SHED also differentiated into endothelial-likecells, as demonstrated by B-galactosidase staining ofcells lining the walls of blood-containing vessels intissues engineered with SHED stably transduced withLacZ. This work suggests that exfoliated deciduousteeth constitute a viable source of stem cells for dentalpulp tissue engineering. (J Endod 2008;34:962–969)

Key WordsAngiogenesis, endodontics, multipotency, odontoblast,scaffold

Regenerative medicine offers exciting opportunities to replace or restore tissues ofthe body after disease and trauma. Tissue engineering approaches aim to fabricate

new replacement body tissues, and such approaches commonly involve seeding of cellsat various stages of differentiation within scaffolds, which can then be implanted (1).The complexity of architecture and function of many tissues, however, provides signif-icant challenges to engineering replacement tissues resembling their physiologic coun-terparts. Use of stem cells, either of embryonic or postnatal derivation, for tissue engi-neering is attractive because it offers greater scope for cell fate to try and mimicphysiologic tissue architecture. However, ethical constraints associated with use ofembryonic stem cells (ESCs) and limitations of readily accessible sources of autologouspostnatal stem cells with multipotentiality pose significant challenges for use of stemcells in tissue engineering. Furthermore, the requirement for good vascularization ofany tissue construct is of paramount importance to its vitality.

The discovery of stem cells in the pulp of permanent teeth (2) and also in decid-uous teeth (3) raised the intriguing possibility of using dental pulp stem cells for tissueengineering (4 – 6). The dental pulp stem cells have been shown to be capable ofself-renewal and multilineage differentiation (7). These stem cells can be isolated frompatients with relatively minimal morbidity, especially when they are retrieved noninva-sively from the pulps of exfoliated deciduous teeth (3). The first successful attempt toengineer complex whole tooth structures used single-cell suspensions dissociated fromporcine third molar tooth buds and suggested the existence of dental pulp stem cells inthis tissue (8). Others have successfully used a similar approach for the bioengineeringof organs for regenerative therapies (9). The concept of using stem cells for dentaltissue engineering was explored by Sharpe and Young (10). They and others demon-strated that it is possible to engineer murine teeth by using adult stem cells of nondentalor dental origin (10 –12). Recently, mesenchymal stem cells isolated from the rootapical papilla of human teeth were shown to be capable of mediating tooth regenerationwith recovery of tooth strength and appearance (13).

Stem cells from human exfoliated deciduous teeth (SHED) have become an at-tractive alternative for dental tissue engineering (3). The use of SHED might bringadvantages for tissue engineering over the use of stem cells from adult human teeth asfollows: (1) SHED were reported to have higher proliferation rate and increase cellpopulation doublings as compared with stem cells from permanent teeth (3). Thismight facilitate the expansion of these cells in vitro before replantation. (2) SHED cellsare retrieved from a tissue that is “disposable” and readily accessible in young patients,ie, exfoliated deciduous teeth. (3) We have previously proposed that dental pulp tissueengineering with stem cells could be ideally suited for young patients who have sufferedpulp necrosis in immature permanent incisors as consequence of trauma (14). Suchtreatment could potentially allow for the completion of vertical and lateral root devel-opment and perhaps improve the long-term outcome of these teeth. The fact that thesepatients are in mixed dentition, and therefore their deciduous molars are at variousdegrees of exfoliation, makes SHED a timely and opportune stem cell source for theengineering of dental pulps in immature permanent teeth.

Although the concept of engineering whole tooth structures offers exciting poten-tial, significant clinical challenges still remain, and engineering or regeneration ofcomponent tissues of the tooth might be a more realistic shorter-term goal. In partic-

From the *Department of Cariology, Restorative Sciences,and Endodontics, University of Michigan School of Dentistry,Ann Arbor, Michigan; †Department of Restorative Sciences,Tokyo Medical and Dental University, Tokyo, Japan; ‡Centerfor Craniofacial Molecular Biology, School of Dentistry, Uni-versity of Southern California; Los Angeles, California; §Unit ofOral Biology, University of Birmingham School of Dentistry;Birmingham, United Kingdom; and �Department of BiomedicalEngineering, University of Michigan College of Engineering;Ann Arbor, Michigan.

Address requests for reprints to Jacques E. Nör, DDS, MS,PhD, Department of Cariology, Restorative Sciences, and End-odontics; School of Dentistry, Department of Biomedical En-gineering; College of Engineering, University of Michigan,1011 N University, Room 2309, Ann Arbor, MI 48109-1078.E-mail address: [email protected]/$0 - see front matter

Copyright © 2008 American Association of Endodontists.doi:10.1016/j.joen.2008.04.009

Basic Research—Biology

962 Cordeiro et al. JOE — Volume 34, Number 8, August 2008

ular, engineering of dental pulp, which is the formative and supportiveorgan for dentin, the major mineralized tissue of the tooth, would pro-vide immense clinical benefits. Dental caries remains the most prevalentinfectious disease of humans, affecting both industrialized and develop-ing nations (15), with consequent economic burden on healthcaresystems globally and quality of life for individuals of all ages. Also, dentaltrauma is common among children and adolescents (16). Dental cariesand trauma to tooth structures frequently lead to the necrosis of thedental pulp tissue and infection, which are typically treated with nonre-generative endodontic approaches, leaving as life-lasting sequelae adevital and weakened tooth.

Key considerations in development of a strategy for pulp tissueengineering include choice of scaffold and cells, which show potential-ity to differentiate into the various cell populations characteristic of thepulp, especially the dentin-secreting odontoblasts and the cells of thevasculature to provide vitality to the tissue. Polymers, such as polygly-colic acid (PGA), have been previously found to act as suitable matricesfor seeding of dental pulp fibroblasts, allowing their proliferation anddevelopment of a tissue with similar cellularity to normal pulp (17).PGA was a more conducive scaffold for dental pulp cell proliferationthan a hydrogel and an alginate (18). Other scaffolds, including a spon-geous collagen, a porous ceramic, and a fibrous titanium mesh, cansupport the attachment, growth, and differentiation of dental pulp stemcells in vitro, and when such constructs were implanted in vivo, the cellsorganized into a well-vascularized tissue that expressed dentin sialo-phosphoprotein, a marker of dentin (19). However, limited extracellu-lar matrix mineralization was observed only with the ceramic scaffold.Our engineering of human blood vessels in immunodeficient mice (20)on the basis of seeding of human dermal microvascular endothelialcells (HDMEC) in porous biodegradable scaffolds and subcutaneoustransplantation in severe combined immunodeficient mice (SCID) of-fers an attractive methodologic approach for pulp tissue engineering.The opportunity to engineer pulp tissue with the required high degree ofvascularity and anastomosis of the engineered blood vessels with thehost vasculature should provide good tissue vitality and enhanced prog-nosis for the tissue constructs.

We demonstrate here the feasibility of engineering a well-vascu-larized pulp-like tissue within 2 weeks of transplantation with humandental pulp stem cells from exfoliated deciduous teeth, drawing on ourrecent report on the maintenance of viability of human tooth slices forseveral weeks after subcutaneous implantation in SCID mice (21).

Materials and MethodsCell Culture

SHED were isolated, cultured, and characterized as described (3).Briefly, SHED were cultured in alpha modification of Eagle medium(Invitrogen, Carlsbad, CA) supplemented with 20% fetal calf serum(Equitech-Bio, Kerrville, TX), 100 �mol/L l-ascorbic acid 2-phosphate(Sigma-Aldrich, St. Louis, MO), 2 mmol/L l-glutamine, 100 units/mLpenicillin, 100 �g/mL streptomycin (Invitrogen) at 37°C in 5% CO2.HDMEC (Cambrex, Walkersville, MD) were cultured in EGM2-MV(Cambrex) as described (20). SHED were transduced with pLAPSNretroviral vector (gift from D. Muller) and selected with Dulbecco mod-ified Eagle medium (Invitrogen) supplemented with 1 mg/mL G418sulfate (Mediatech, Herndon, VA) to generate stable clones expressingLacZ. SHED stably transduced with pLXSN were generated as emptyvector controls as described (22).

Tooth Slice/Scaffold PreparationThirty noncarious freshly extracted third molars were collected

from the Oral Surgery Clinic at the University of Michigan from 15- to

22-year-old patients with informed consent and institutional reviewboard approval. Residual soft tissues were removed with a scalpel, andthe dental surfaces were wiped down with 70% ethanol. The teeth werethen transversally cut at the cervical region with a diamond-edged bladeat low speed under cooling with sterile phosphate-buffered saline (1�PBS; Mediatech, Inc) to obtain slices of approximately 1-mm-thickness(Fig. 1A), with the volume of the dental pulp chambers ranging between29 – 43 mm3, as previously described (21). The pulp tissue was re-moved with forceps without touching the dentinal walls to prevent theremoval of the predentin layer (Fig. 1A). The pulp cavity of each toothslice was filled with sieved sodium chloride (250 – 450 �m) and poly-L-lactic acid (PLLA) (molecular weight, 250,000 g/mol; BoehringerIngelheim, Germany) dissolved with chloroform as described (20).After polymerization of PLLA, the salt was completely removed withdistilled water. The day before the implantations, the tooth slice/scaf-folds were hydrophilized with sequential incubations (5 minutes) inethanol (100%, 90%, 80%, and 70%) and washed with sterile 1� PBSovernight at 4°C as described (20). A minimum of 6 tooth slices/scaf-folds per experimental group was evaluated at each time point. At least3 independent experiments were performed to verify reproducibility ofresults for each data set presented here.

Cell Seeding and Transplantation of Tooth Slice/Scaffolds inImmunodeficient Mice

Tooth slice/scaffolds were divided into 3 groups: (1) 8 � 105

SHED cells alone; (2) 0.8 � 105 SHED � 7.2 � 105 HDMEC; or (3)Control, empty scaffolds (no cells). Alternatively, SHED-LacZ or SHED-LXSN was seeded in the tooth slice/scaffolds to evaluate the fate of thecells transplanted. Cells were resuspended in a 1:1 mixture of Matrigel(BD Biosciences, Bedford, MA) and cell culture medium (total volumeof 18 �L per scaffold) to allow for their seeding in the scaffolds. Thetooth slice/scaffolds containing cells were incubated for 30 minutes at37°C to allow for the setting of the Matrigel as described (20). Controlscaffolds received 36 �L of a 1:1 mixture of Matrigel and cell culturemedium without cells. The tooth slice/scaffolds were implanted bilater-ally in the subcutaneous tissue of the dorsum of 5- to 7-week-old maleimmunodeficient mice (CB.17 SCID; Charles River, Wilmington, MA)(Fig. 1A). After 14 –28 days, the implants were retrieved, fixed in 10%buffered formalin at 4°C for 24 hours, demineralized with 10% formicacid at 4°C until the dentin offered no resistance to cutting with a blade(5– 8 days), and then processed for histology. Histologic sections (5-�m-thick) were stained with hematoxylin-eosin or kept unstained forimmunohistochemistry. In addition, tooth slices were prepared from 3healthy third molars, but their pulps were not removed, as described(21). These tooth slices served as additional controls for our experi-ments.

ImmunohistochemistrySections were deparaffinized in xylene, rehydrated, washed in

TBS-T, pH 8.0, and then incubated in antigen retrieval solution (Dako-cytomation; Dako, Carpinteria, CA) for 20 minutes at 90°C–95°C. A1:100 dilution of a polyclonal rabbit anti-human Factor VIII antibody(Lab Vision, Fremont, CA) was used to localize the microvascular net-works formed by the implanted human endothelial cells. Color devel-opment was performed with a Dako EnVision � system kit (AEC, Da-kocytomation) according to the manufacturer’s instructions. Thenumber of microvessels in 10 random fields per implant was countedunder a light microscope (100�) in 6 implants from independent miceper condition. For dentin sialoprotein (DSP) staining, we used Trypsin(Lab Vision) for antigen retrieval and a 1:100 dilution of the anti-humanDSP polyclonal antibody (C-20; Santa Cruz Technology, Santa Cruz, CA).Color was developed with a DAB solution (Vector Laboratories, Inc,


JOE — Volume 34, Number 8, August 2008 Dental Pulp Tissue Engineering with Stem Cells 963

Burlingame, CA). Tissue sections were counterstained with hematoxy-lin, and the cover slides were mounted by using an aqua-poly mountingsolution (Polysciences, Inc, Warrington, PA). As controls, we stainedsections with an isotype-matched nonspecific immunoglobulin G (SantaCruz Technology). The number of cells lining the dentin in 10 randomfields per implant was counted under a light microscope (400�) in 6implants from independent mice per condition.

B-Galactosidase StainingImmediately after retrieval from the SCID mice, tooth slice/scaf-

folds seeded with SHED-LacZ or SHED-LXSN cells (alone or combinedwith HDMEC) were immersed in washing buffer (0.1 mol/L NaPO3 [pH8.3], 2 mmol/L MgCl2, 0.1% sodium deoxycholate, 0.02% TritonX-100) and stained at 4°C in the dark with the staining solution (1mg/mL X-gal, 5 mmol/L K3Fe(CN)6, 5 mmol/L K4Fe(CN)6-3H2O). Nextday, implants were rinsed thoroughly in H2O, fixed in 10% neutral-buffered formalin for 1 hour, and processed for histology. After depar-affinization, the slides were stained with 0.1% safranin O for 3 minutes,rinsed briefly in H2O, dehydrated in 95% followed by 100% ethanol for30 seconds each, and cleared in xylene for 30 seconds before mounting.

Transmission Electron MicroscopyTo analyze the ultrastructure of the engineered cells lining the

predentin, 5-�m-thick sections from formalin-fixed, paraffin-embed-ded specimens were deparaffinized, rehydrated in graded ethanol se-

ries, and embedded in epoxy resin. Briefly, the tissue sections werere-fixed in 0.5% glutaraldehyde, post-fixed with 1% OsO4, dehydratedthrough a graded series of ethanol, and embedded in Epon 812. Ultra-thin sections (60- to 80-nm-thick) were cut with an ultramicrotome(Reichert Ultracut-N; Reichert-Nissei, Tokyo, Japan) and examined in atransmission electron microscope (H7100; Hitachi, Tokyo, Japan), asdescribed (23).

Statistical AnalysesDescriptive analysis, paired t tests, and one-way analysis of variance

followed by Tukey test were performed by using Sigmastat 2.0 software(SPSS, Chicago, IL). The significance level was determined at P � .05.

ResultsThe overall strategy for dental pulp tissue engineering presented

here consists of the manufacturing of porous biodegradable scaffolds inthe root canal of extracted human teeth followed by the seeding of SHEDin the scaffolds and transplantation of the tooth slice/scaffold– contain-ing cells into the subcutaneous tissue of immunodeficient mice (Fig.1A). The PLLA scaffolds occupied the space of the pulp chamber in thetooth slices and were in close contact with surrounding predentin (Fig.1B, C). After a period of 14 –28 days, we observed a dental pulp-liketissue with morphologic characteristics that resemble those of a normaldental pulp (Fig. 1D, E).

Figure 1. Engineering of a dental pulp tissue with dental pulp stem cells. (A) Schematic diagram of strategy for dental pulp tissue engineering. (B) Biodegradablescaffold is prepared within the root canal and then seeded with dental pulp stem cells only, or dental pulp stem cells mixed with endothelial cells. Tooth slice containingcells is then implanted in the subcutaneous tissue of immunodeficient mice. (C) High magnification of the tooth slice/scaffold showing the interface between scaffoldand predentin. (D) Low magnification (100�) of a dental pulp engineered with SHED and primary HDMEC, 14 days after implantation in an immunodeficient mouse.(E) High magnification (400�) of the boxed area of the engineered dental pulp presented in (D).



We have seeded either SHED alone or SHED and HDMEC in thetooth slice/scaffold devices. As controls, we implanted tooth slice/scaf-folds without cells. To begin morphologic characterization of the engi-neered dental pulps, we counted the number of cells lining the preden-tin (Fig. 2A–G). Although a higher cell density and tissue organizationwere observed throughout the whole engineered tissue in the SHED �

HDMEC group, we did not observe a significant effect on the density of cellslining the predentin when dental pulps were engineered with SHED alone orSHED plus HDMEC (Fig. 2A). However, there was significantly higher num-ber of cells lining the predentin in the pulp-like tissues engineered withSHED and HDMEC or SHED alone, as compared with the tissues resultingfrom the implantation of empty scaffolds (P � .03) (Fig. 2A).

Figure 3. SHED cells differentiate into odontoblast-like cells after transplantation in tooth slice/scaffolds. (A) Dental pulp engineered with SHED only and immuno-stained with anti-human DSP antibody. (B) Dental pulp engineered with SHED � HDMEC. (C) Dental pulp from a control tooth that was extracted for orthodonticreasons. (D) Dental pulp engineered with SHED � HDMEC immunostained with an isotype-matched nonspecific immunoglobulin as control. DAB system was usedto generate the color (DSP-positive cells stain dark brown). Three independent experiments were performed to verify reproducibility of results. All images are at400�.

Figure 2. Histologic evaluation of the cellularity of the engineered dental pulps. (A) Graph depicting the number of cells lining the predentin of engineered dentalpulps in tooth slice/scaffolds seeded with SHED cells alone, SHED and HDMEC, or empty scaffold controls (*P � .05). Cell counts were performed at 400� in 10random fields from 6 implants per experimental condition. Three independent experiments were performed to verify reproducibility of results. (B–D) Lowmagnification (200�) and (E–G) high magnification (400�) of representative microscopic fields of engineered dental pulps. Tissues were stained withhematoxylin-eosin.



To further evaluate the characteristics of the engineered pulp, weperformed immunohistochemistry with DSP, one of the accepted mark-ers of odontoblast differentiation (Fig. 3). The cells adjacent to thepredentin in the engineered pulps (Fig. 3A, B) stained positive for DSP,suggesting their differentiation into odontoblast-like cells. Notably, odonto-blast-like cells in the SHED plus HDMEC group showed stronger immuno-reactivity for DSP than the same cells in the tooth slice/scaffolds seeded onlywith SHED. As positive controls, we stained the pulp tissue of a freshlyextracted tooth (Fig. 3C), and our negative controls were tissue sectionsstained with an isotype-matched irrelevant antibody (Fig. 3D).

Transmission electron microscopy was performed to evaluate theultrastructure of the cells adjacent to the predentin in engineered dentalpulps. These cells showed morphologic characteristics that resembledthose of odontoblast cells, including the eccentric polarized position of

the nucleus at the basal part of the cell body, and the observation ofseveral gap junctions at the cell contact area (Fig. 4). Notably, well-developed rough endoplasmic reticula, a well-developed Golgi’s com-plex, and numerous vesicles could be found (Fig. 4A), suggesting thatthese cells had high secretory activity (typical of odontoblasts).

To evaluate the microvessel density of the engineered dental pulps,we performed immunohistochemistry with an anti-human Factor VIIIantibody. We observed an increase in microvessel density when SHEDalone or SHED plus HDMEC cells were seeded in the tooth slice/scaf-folds and implanted in the mice, as compared with the empty scaffoldcontrols (P � .001) (Fig. 5). However, the co-implantation of SHEDand HDMEC cells did not result in a significant increase in microvesseldensity, when compared with the implantation of SHED alone (Fig.5A–C).

Figure 4. SHED cells acquire ultrastructure features of odontoblastic cells after transplantation in the tooth slice/scaffold devices. (A) Electron micrographs of thecells lining the predentin 21 days after implantation of SHED and HDMEC in the tooth slice/scaffold. Cells presented nuclear polarization, ie, nucleus was positionedin the extremity of the cell that was away from the predentin. Bar � 4 �m. (B) Higher magnification of the small boxed area in (A). A gap-junction is observed between2 adjacent cells. (C) Higher magnification of the large boxed area in (A). Photomicrograph depicting well-developed rough endoplasmic reticula (rER), mitochon-dria (Mt), and Golgi’s apparatus (G). (D) Electron micrographs of 2 odontoblast-like cells in a dental pulp engineered with SHED only. rER are less developed, ascompared with the odontoblast-like cells when both HDMEC and SHED were implanted. Bar � 5 �m. (E) Higher magnification of the boxed area in (D). Arrowheadsdepict cell-to-cell contacts with several gap-junctions.



To evaluate the fate of the dental pulp stem cells after transplanta-tion, we stably transduced SHED with LacZ (Fig. 6A). We observedLacZ-positive cells in the area adjacent to the predentin (Fig. 6D).Interestingly, we have also found innumerous functional blood vesselslined with LacZ-positive cells (Fig. 6B, D). Indeed, the majority (nearly100%) of the blood vessels found in the pulps engineered with SHED-LacZ stained positive for LacZ and contained blood cells in their lumen,which demonstrates that the transplanted dental pulp stem cells werecapable of differentiating into blood vessels that anastomosed with thehost vasculature.

DiscussionWe describe a novel method for the engineering of a highly vascu-

lar connective tissue showing an architecture and histologic structureclosely resembling those of its physiologic counterpart. This methodoffers exciting opportunities for regeneration of dental pulps, one of themost commonly injured/diseased tissues of the body, with consequentpotential for impact on healthcare and the possibility for application ofthe method to other body tissues such as bone (24). The use of multi-potent postnatal stem cells from a disposable source (ie, exfoliateddeciduous teeth) that can be readily isolated noninvasively and thatretain their potentiality after in vitro expansion offers significant advan-tages. Importantly, these cells form a functional vasculature as well asconnective tissue secreting cells of the soft and hard tissues of the tooth.

The SHED cells (3) used here are capable of self-renewal andshow multipotentiality (7). They have been the focus of commercialstem cell banks seeking autologous stem cell sources derived noninva-

sively for application in a variety of therapies. The ability of these cells todifferentiate into the various populations found in the dental pulp con-trasts with our control (empty) scaffolds, which, although populated byinfiltration of host cells, showed very different cellular structure to thetissues resulting from implantation of dental pulp stem (SHED) cells.They showed low cellularity, poor spatial organization, and low mi-crovessel density. In the SHED cell–seeded tissue constructs, the cells atthe periphery of the tissue showed characteristics of active dentin-se-creting odontoblasts, including expression of DSP (25) and ultrastruc-tural characteristics of nuclear polarization, the presence and positionof cell-cell gap junctions, and a well-developed endoplasmic reticulum(26). It is known that DSP is not a specific marker for odontoblast cells,because it can also be expressed by osteoblasts (27). However, DSP’sexpression level is approximately 400-fold higher in dentin as com-pared with bone (27), suggesting that it is highly expressed in odonto-blast cells. Together, the DSP expression assays and the anatomic anal-ysis performed here led us to conclude that SHED seeded in toothslice/scaffolds were capable of differentiating into odontoblast-likecells, as has been previously described (3). Importantly, the SHED cellsappeared capable of forming a microvascular network, which is a pre-requisite for the successful engineering of most tissues and organs (28,29). To expedite the organization of the microvascular network in ourpulp tissue constructs, we co-implanted SHED cells and primary humanendothelial cells. When SHED cells were co-implanted with human en-dothelial cells, the resulting pulp tissue constructs had better organiza-tion and greater cellularity than when SHED cells alone were implanted.These findings suggest that co-implantation of HDMEC provides for the

Figure 5. Co-implantation of endothelial cells did not enhance the microvascular density of the dental pulps engineered with SHED. (A) Graph depicting total numberof microvessels in tooth slice/scaffolds seeded with SHED cells alone, SHED plus HDMEC, or empty scaffold controls (*P � .05), as determined by Factor VIII–positivecells surrounding a lumen. (B) Dental pulp engineered with SHED only and immunostained with anti-human Factor VIII antibody. (C) Dental pulp engineered withSHED plus HDMEC. (D) Dental pulp from an empty scaffold control. AEC system was used to generate the color (Factor VIII–positive cells stain in red). Microvesselcounts were performed at 100� in 10 random fields from 6 implants per condition. Three independent experiments were performed to verify reproducibility ofresults. All images are at 200�.



quick organization of a microvascular network and influx of oxygen andnutrients to the SHED, improving their survival after transplantation andenhanced tissue cellularity. Surprisingly, however, we did not find sig-nificant differences in the number of blood vessels in these 2 experi-mental conditions after a period of 14 –28 days. This might reflect thefact that SHED differentiated into blood vessel–forming cells. This hy-pothesis is supported by our finding that SHED-LacZ cells were capableof organizing functional (blood carrying) blood vessels. Alternatively,the lack of a significant difference between the 2 experimental condi-tions might reflect the fact that these tissues have achieved vascularhomeostasis by 14 –28 days, and therefore the number of blood vesselsrequired for adequate tissue oxygenation is similar in both conditions.These hypotheses are currently under active investigation in our labo-ratory. Remarkably, the stem cells were not only able to form the vas-cular structures but also to connect them (anastomose) with the mousevasculature. Human embryonic stem cells have been shown to be capa-ble of differentiating into endothelial cells (28), and dental pulp stemcells were reported to have differentiation potential into osteoblasts andendotheliocytes (30). However, to our knowledge such differentiationpotential has not been previously reported for dental stem cells in thecontext of dental pulp tissue engineering. Taken together, these resultssuggest that the co-implantation of endothelial cells might not be anecessary step for the engineering of tissues with SHED.

Differentiation of SHED to form dentin-secreting odontoblasts, fi-broblasts, and the microvascular network in the tissue construct re-quires morphogenetic signaling, and the choice of scaffold and its modeof presentation were likely important to this. Dentin matrix contains acocktail of sequestrated growth factors and bioactive molecules (31),and solubilization of the matrix allows for their release and potentialregenerative effects on undifferentiated cells (32) and in reparative

dentin formation (33–35). We hypothesize that the low pH generatedlocally by the degradation of the PLLA-based scaffold that we have usedhere might have contributed to the mobilization of these proteins. In-deed, we observed that most of these scaffolds are degraded within theexperimental period used here of 2– 4 weeks, as previously described(20). Although such scaffolds are constrained by the need to cast theshape and size of the tissue construct, acid-modified hydrogels alsoappear to have similar morphogenetic capacity (36) and might facilitatedevelopment of injectable scaffolds for applications in tissues with ac-cess constraints. Thus, the combination of the scaffold and its presen-tation within the slice of tooth matrix containing morphogenetic signal-ing molecules are important features of the methodology reported.

In conclusion, this methodology allows for the engineering of den-tal pulp tissue constructs resembling their physiologic counterparts onthe basis of seeding of a simple scaffold with cells readily isolated non-invasively and presented within a slice of acellular tooth matrix contain-ing endogenous sequestrated morphogenetic signals. Here we used1-mm-thick slices to begin to understand the potential of SHED for theengineering of dental pulps, as well as the need of co-implantation ofendothelial cells for successful vascularization of this tissue. It is clearthat the translation of such strategy to the clinic will require the ability toengineer pulps within the entire length of the root canal. Nevertheless,the authors firmly believe that SHED have the potential to significantlyimpact oral healthcare and might also have broader applications to theengineering of other body tissues.

AcknowledgmentsThe authors would like to thank Chris Yung for the drawing of

the model and Chris Strayhorn for the histologic preparation of the

Figure 6. Fate of SHED cells after transplantation. (A) �-galactosidase staining of SHED-LacZ cells in vitro (200�). (B) �-galactosidase staining of a dental pulpengineered with SHED-LacZ cells (400�). Arrow points to �-galactosidase–positive blood vessel. (C, D) Immunohistochemistry with anti-LacZ antibody (400�).(C) Control dental pulp engineered with SHED-LXSN (empty vector control). (D) Dental pulp engineered with SHED-LacZ. Arrow points to LacZ-positive blood vessel,and arrowheads point to LacZ-positive cells lining the predentin. Images for �-galactosidase staining and immunohistochemistry were not counterstained.



samples. The authors want to thank Tatiana Botero, Kathleen Neiva,Flavio Demarco, and Eoin Mullane for critical review and valuablesuggestions to this manuscript. This study was supported by theDepartment of Cariology, Restorative Sciences, and Endodontics,University of Michigan School of Dentistry (J.E.N.) and by Grants-in-Aid for Scientific Research (Grant #18791393) from the JapanSociety for the Promotion of Sciences (T.K.).

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dental pulp tissue engineering with stem cells from exfoliated deciduous teeth

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