Autologous Dental Pulp Stem Cells in Regeneration ofDefect Created in Canine Periodontal TissueAfshin Khorsand, DMD, MS1

Mohamadreza Baghaban Eslaminejad, PhD2*Mohadeseh Arabsolghar, DMD, MS3

Mojghan Paknejad, DMD, MS1

Baharak Ghaedi, DMD, MS4

Amir Reza Rokn, DMD, MS1

Neda Moslemi, DMD, MS1

Hamid Nazarian, MSc2

Shahrbanoo Jahangir, MSc2

This study aimed to investigate effects of dental pulp stem cells (DPSCs) on regeneration of a defect experimentally

created in the periodontium of a canine model. Surgically created mesial 3-walled periodontal defects with ligature-

induced periodontitis were produced bilaterally in the first lower premolar teeth of 10 mongrel dogs. Simultaneously,

DPSCs were derived from the maxillary premolar teeth of the same dogs. Four weeks after creation of the

periodontitis model, autologous passaged-3 DPSCs combined with Bio-Oss were implanted on one side as the test

group. On the other side, only Bio-Oss was implanted as a control. Eight weeks after surgery, regeneration of the

periodontal defects was evaluated histologically and histomorphometrically in terms of bone, periodontal ligament

(PDL), and cement formation. Histologically, in all test specimens (10 defects), regeneration of cementum, bone, and

PDL was observed. In the control groups, although we observed the regeneration of bone in all defects, the formation

of cementum was seen in 9 defects and PDL was seen in 8 defects. Histomorphometric analyses showed that the

amount of regenerated cementum and PDL in the test groups (3.83 6 1.32 mm and 3.30 6 1.12 mm, respectively)

was significantly higher than that of the control groups (2.42 6 1.40 mm and 1.7761.27 mm, respectively; P , .05). A

biocomplex consisting of DPSCs and Bio-Oss would be promising in regeneration of periodontal tissues.

Key Words: dental pulp stem cells, regeneration, periodontal defect, Bio-Oss

INTRODUCTION

Periodontitis is a complex disease of the

periodontium that results in consider-

able defects to alveolar bone, gingival

tissue, and periodontal ligament (PDL).1

Reconstruction of defects resulting from

periodontal disease is a major challenge to regen-

erative medicine in the oromaxillofacial field. On the

other hand, in the field of tissue regeneration, the

use of tissue-resident stem cells as regenerative

materials has gained considerable attention owing

to their self-renewal property and multilineage

differentiation capacity.

Among stem cells, dental stem cells are of

interest as they are easily accessible and can be

collected noninvasively with low morbidity.2 In this

context, the periodontal ligament stem cell (PDLSC)

has been successfully investigated in some animal

models. Liu et al3 investigated the utility of

autologous PDLSC to treat ligature-induced peri-

odontitis in miniature swine. The 3-walled defect

was generated in the first molar area of miniature

1 Dental Research Center, Periodontics Department, Faculty ofDentistry, Tehran University of Medical Sciences, Tehran, Iran.2 Department of Stem Cells and Developmental Biology at CellScience Research Center, Royan Institute for Stem Cell Biologyand Technology, ACECR, Tehran, Iran.3 Department of Periodontology, School of Dentistry, KermanUniversity of Medical Sciences. Kerman, Iran.4 Department of Periodontology, School of Dentistry, HamedanUniversity of Medical Sciences. Hamedan, Iran.* Corresponding author, e-mail: eslami@royaninstitute.orgDOI: 10.1563/AAID-JOI-D-12-00027

Journal of Oral Implantology 433

RESEARCH

swine, and autologous PDLSCs obtained fromextracted teeth were transplanted into the surgi-cally created periodontal defect areas.3 Akizuki et al4

performed a pilot study in beagle canine teeth toinvestigate periodontal regeneration by use ofPDLSC in dehiscence defects that were surgicallycreated on the buccal surface of the canine firstmolar. Autologous periodontal ligament (PDL) cellsobtained from extracted canine premolars wereinduced to form sheets when a temperature-responsive cell culture dish was used. The sheetwith a reinforced hyaluronic acid carrier was thenapplied to the defects.4 In another study, Dogan etal5 researched PDL-derived stem cells for theregeneration of periodontal tissue in the caninemodel by seeding fibroblast-like cells derived fromregenerated PDL in artificial furcation defects.

A few studies have researched the regenerativepotential of dental pulp stem cells (DPSCs), whichwere first isolated by Gronthos et al,6 They describedDPSCs as colonogenic cells that were capable ofproducing osteoblastic and chondrocytic cells in vitroand odontoblastic cells in vivo. The followinginvestigations have indicated the broad differentia-tion capacity of the pulp-derived cells, which includedtheir ability to give rise to neural and endothelial celllineages.6–9 In addition, some studies have indicatedthat DPSCs are capable of forming well-vascularizedlamellar bone after grafting.2,9,10 For example, deMendonca Costa et al11 created 2 symmetric full-thickness cranial defects (5 3 8 mm in dimension) onthe rat parietal region and attempted to reconstructthe defects with collagen membrane and DPSCs.

The aim of the present study is to examine thepotential of DPSCs combined with biomaterials topromote regeneration of periodontium after experi-mentally created periodontitis is induced in caninemodels. Dogs are appropriate models because theirperiodontal tissues and tooth size are quite similar tothose of humans. In addition, they often develop earlydental plaque, which has many structural similaritiesto that occurring in humans. Furthermore, periodon-tal diseases can be easily induced in dogs.12–16

Although subgingival plaque formation in dogs maynot develop identically to that in humans, dogs maystill serve as a conventional model for investigation.

The bone regenerative capacity of DPSCs hasbeen investigated previously17–19; however, to dateno investigation has evaluated DPSCs in theregeneration of periodontium that consisted of

cementum, PDL, and supporting bone. The scaffoldused in this study was xenograft, a natural bovinebone mineral with osteoconductive properties andhigh biocompatibility. This biomaterial has beentested in multiple randomized clinical trials andregistered in the Cochrane Library.20

MATERIALS AND METHODS

Animals

Ten male mongrel dogs with healthy periodontium(1–2 years old, weighing 14–22 kg) were obtainedfrom the Institute of Animal Science of Tehran 2weeks before the beginning of the investigation.The dogs were kept under conventional conditions,vaccinated, and treated with antifungal drugs.Animal were fed soft food (Friskies, Purina, MarneLa Vallee, France).

Cells

Based on our pilot study, we decided to extract 2maxillary premolar teeth from each dog for DPSCs.Teeth were kept in Dulbecco’s modified eaglemedium (DMEM, Gibco, Paisley, UK) and quickly sentto the laboratory for cell culture. At the Royan InstituteCell Culture laboratory, tooth surfaces were cutaround the root-enamel boundary using dentalfissure burs. Pulp tissues were then gently collectedfrom the chambers and subjected to enzymaticdigestion using a solution of 3 mg/mL collagenasetype I (Sigma, St Louis, Mo) and 4 mg/mL dispase(Sigma) for 30 minutes at 378C. The digest wasprovided with about 3 mL DMEM supplemented with10% fetal bovine serum (FBS; Gibco) and centrifugedat 1200 rpm for 5 minutes. A single-cell suspensionwas then prepared and cells were plated at 103 cells/well in 6-well culture plates in an atmosphere of 5%CO2 and 378C. The culture medium was changedtwice weekly until confluency was achieved. Conflu-ent cultures were passaged at 1:3 ratios until sufficientcells became available for additional experimentation.

Flow cytometry

Flow cytometric analysis was used to characterizethe isolated cells with respect to their surfaceantigen profile. About 1.5 3 105 passaged-3 cellswere suspended in 100 lL of phosphate bufferedsaline (PBS) in 5 mL tubes that contained 5 lL of thefollowing fluorescein isothiocyanate–conjugated

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DPSCs in Regeneration of Periodontal Tissue

antibodies: CD146, CD44, CD90, SSEA-4, and anti-macrophage (Becton Dickinson, Franklin Lakes, NJ)followed by incubation at 48C for 30 minutes in adark room. The solution was then centrifuged at1200 rpm for 4 minutes. The cells were dispersed in300–500 lL washing buffer and then analyzed byflow cytometry (FACSCalibur cytometer equippedwith 488 nm argon lasers, BD Bioscience, San Jose,Calif). The isotope controls were IgG 2 and IgG 1.WinMDI software (Version 2.9; Purdue University,West Lafayette, Ind) was used to analyze the flowcytometric results.

Multilineage differentiation

Osteogenesis

Passaged-3 cells were plated in 6-well culture platesuntil confluency. Then, the proliferation mediumwas replaced by induction medium that includedDMEM supplemented with 50 lg/mL ascorbic acid2-phosphate, 10 nm dexamethasone, and 10 lMbeta glycerol phosphate (Sigma). The differentiationculture was maintained for 21 days, during whichcultures were fed twice weekly. Alizarin red (Sigma)staining as well as reverse transcriptase-polymerasechain reaction (RT-PCR) analysis was used toexamine differentiation.

Adipogenesis

Passaged-3 cells were cultivated in 6-well cultureplates until confluency. The medium was thensubstituted with differentiation medium that con-sisted of DMEM supplemented with 100 nmdexamethasone and 50 lg/mL indometacin (Sig-ma). After 21 days of incubation and twice-weeklymedium replacement, differentiation was evaluatedwith oil red staining in addition to RT-PCR analysis.

Chondrogenesis

The micro-mass culture technique was used topromote chondrogenic differentiation of isolatedcells. Passaged-3 cells were suspended in 5 mLDMEM medium in 15 mL tubes and centrifuged at1200 rpm for 5 minutes. The supernatant wasremoved and chondrogenic medium was addedonto the cell pellet. The chondrogenic mediumconsisted of DMEM supplemented with 3 ngtransforming growth factor beta, 10 ng bonemorphogenetic protein-6, 50 mg/mL transferrin-selenium-insulin, 50 mg bovine serum albumin, and1% FBS (all from Gibco). The culture was maintained

at 378C and 5% CO2 for 21 days, after which theculture was prepared for light microscopy. Sections5 lm thick were created and stained with toluidineblue. The culture was analyzed by RT-PCR forexpression of cartilage specific genes.

RT-PCR analysis of gene expression

Cellular differentiation potential was determined byRT-PCR detection of specific gene expression. TotalRNA was collected from the cells induced todifferentiate along bone, cartilage, and adipose celllineages, as described previously, using RNX-Plus.solution (CinnaGen Inc, Tehran, Iran). Before reversetranscription, RNA samples were treated with DNase I(Fermentas, Opelstrasse, Germany) to remove con-taminating genomic DNA. The standard reverse-transcription reaction was performed with 5 lg totalRNA using Oligo (dT) 18 as a primer and theRevertAid H Minus First Strand cDNA Synthesis Kit(Fermentas, Opelstrasse, Germany) based on themanufacturer’s instructions. Reaction mixtures forpolymerase chain reaction (PCR) included 2.5 lLcDNA, 13 PCR buffer (AMS, CinnaGen, Tehran, Iran),200 lM dNTPs, 0.5 lM of each primer pair (Table 1)and 1 unit/25 ll reaction Taq DNA polymerase(Fermentas). Each PCR was performed in triplicateand under linear conditions. The products wereanalyzed on 2% agarose gel and visualized byethidium bromide staining.

Scaffold

In this study Bio-Oss granules were purchased(Geistlich, Osteohealth Biomaterials, Bern, Switzer-land) and used. Measurements of the crystalline sizeof Bio-Oss have shown the same characteristics asthe tiny crystal size observed in clinically normalhuman bone. The spongiosa structure indicates aninterconnected pore system (300–1500 lm) with acrystalline size of 10–60 nm.

Cell loading

To load the cells, 3 or 4 Bio-Oss granules were placedin small wells and 2 3 107 autologous passaged-3DPSCs were placed on the top surface of thescaffolds. The cultures were placed in an incubatorat 378C. At this time, the cells penetrated into thescaffold porosity and attached to the ceramicsurfaces. Two hours after loading initiation, con-structs were used for implantation. To calculate theamount of cells successfully loaded into the scaffold

Journal of Oral Implantology 435

Khorsand et al

pores, all cells that appeared within the wells, either

floating or adhered, were collected and counted with

a hemocytometer. To ensure that DPSCs were within

the scaffold surfaces, the loaded Bio-Oss were fixed

in 10% formaldehyde in PBS buffer, decalcified in

10% ethylenediamine tetra-acetic acid for 24 hours,

and processed for light microscopic observation.

Creation of a periodontitis model

To create periodontitis, the method of Liu et al3 wasused. In brief, dogs were anesthetized with intra-muscular injections of a combination of ketamine (8mg/kg) and diazepam (0.5 mg/kg). Then, a crestalincision was made from the mandibular canine tothe midbuccal of the first premolar and the

TABLE 1

Primers used in reverse transcriptase-polymerase chain reaction

Genes Primer Sequences (50-30) Annealing Temperature (8C) Length (bp) Gene Bank Code

Col IA1 F: 50 tca cct acc act gca aga ac 30 62 302 NM_001003090

R: 50 agt tta cag gaa gca gac agg 30

COL II F: 50 caa gaa cag cat tgc cta cc 30 57 550 NM_001006951

R: 50 agt tag ttt cct gcc tct gc 30

LPL F: 50 gtg aac atg tgt ggg tat ctg 65 370 XM_534584.2

R: 50 cta ggg cct tta ctg act ggaAggrecan F: 50 aca gga ttg aag tca gtg gag 30 63 527 U65989

R: 50 gtt gac aaa ctc ctg ttc ctc 30

GAPDH F: 50 cca cgg caa att cca cgg cac ag 30 57 652 NM_001003142

R: 50 ggg gtc cct ccg atg cct gct tc 3Osteopontin F: 50 acg atg tag atg aag atg atg g 30 57 548 XM_535649

R: 50 gct ttg act taa ttg gct gac 30

PPARG 2 F: 50 atc cct ctt cca tgc tgt tat g 30 62 253 AJ972913

R: 50 ata gtg tgg agt gga aat gct g 30

FIGURE 1. Creation of periodontitis and implantation of biomaterial. (a) Photograph of surgical defect in the mesial portion ofthe dog’s lower premolar. (b) Ligature around the cervical portion of the tooth for the creation of periodontitis. (c) Secondphase of surgery: Implantation of biomaterial was performed 4 weeks after creation of the model. (d) Suturing with 3-0nylon thread.

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DPSCs in Regeneration of Periodontal Tissue

mucoperiosteal flap was elevated. Alveolar bone

was removed by a surgical bur, and a surgical defect

was created bilaterally in the mesial of the mandib-

ular first premolar (Figure 1a). The surgical defect

was 3 mm buccolingually, 5 mm apico-coronally,

and 8 mm mesiodistally. The surface of the root was

removed by a round bur to ensure removal of the

cementum and PDL. Two notches, one at the level of

the bone crest and the other on the floor of the

defect, were made on the root surface by a round

bur. In total, 20 defects were created in 10 dogs. A 3-

0 silk thread was used around the cervical region of

the first premolars to accelerate the accumulation of

microbial plaque (Figure 1b).

Transplantation

Four weeks after creation of the model, signs of

periodontitis, such as bleeding on probing, pocket

formation, and loss of attachment, were observed.

At this time under general anesthesia, the teeth

were cleaned, the full-thickness flap was raised, and

granulation tissue was removed from the defect by

curettes. Then, scaling and root planning was

performed to decontaminate the root surface. The

FIGURE 2. Pulp-derived cell culture. (a) A large colony formed at the primary culture, day 5. Bar ¼ 200 lm; originalmagnification, 330. (b) Confluent culture of pulp-derived cell culture. Bar¼ 200 lm; original magnification, 350 (c–g) Flowcytometric analysis of passaged-3 canine pulp-derived stem cells. Most of the cells expressed CD90 and CD44 surfaceantigens, and CD146, SSEA-4, and anti-macrophage were expressed on a small percentage of the cells.

Journal of Oral Implantology 437

Khorsand et al

defects were randomly divided into 2 groups. On

one side, about 3 or 4 Bio-Oss granules (1–2 mm)

were implanted, whereas on the other side, the

same amount of Bio-Oss granules loaded with

autologous passaged-3 DPSCs were grafted (Figure

1c). The flap was advanced and sutured by the use

of 3-0 nylon thread (Figure 1d). All animals received

cefazolin (22 mg/kg, every 12 hours for 3 days) and

tramadol (2 mg/kg, every 12 hours for 3 days)

intramuscularly. A mouthwash of chlorhexidine

digluconate was administered by a cotton roll twice

daily for 2 weeks at the surgical sites.

Histologic and morphometric evaluation

Eight weeks after transplantation, all animals were

killed by an overdose of sodium thiopental. Samples

of the first premolars with mesial defects were

harvested, fixed with 10% buffered formalin, decal-

cified in 15% nitric acid, and embedded in paraffin.

About 9 sections were cut: the first section was cut

FIGURE 3. Multilineage differentiation potential of canine pulp-derived stem cells. Passaged-3 cells tended to differentiatealong bone (a–b, bar ¼ 50 lm), adipose (c–d, bar ¼ 50 lm), and cartilage (e–f, bar ¼ 200 lm) cell lineages.

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DPSCs in Regeneration of Periodontal Tissue

at the mid-buccolingual aspect of the defects and

then every section was cut bilateral from the first

section at 5 lm thicknesses, stained with hematox-

ylin and eosin, and evaluated by light microscope

(Olympus D 25, Olympus, Tokyo, Japan). Each

section was analyzed quantitatively and histomor-

phometrically by analysis software (Olympus, ver-

sion 3.2) according to the following parameters: (1)

cementum formation, that is, the length of cemen-

tum formed between 2 notches; (2) alveolar bone

formation, that is, the length of bone formed

between 2 notches; and (3) PDL formation, that is,

the length of PDL formed between 2 notches.

Statistical analysis

Groups were compared using the Wilcoxon signed

ranks test. Statistical analysis was considered

significant at P , .05.

RESULTS

Cell cultures

The primary cultures established from dental pulp

cells contained multiple colonies that consisted of

fibroblastic cells (Figure 2a). A number of small clear

cells were also present among the fibroblastic cells.

The primary culture reached confluency in 10 days

as the colonies grew (Figure 2b). In the subcultures

cells tended to proliferate rapidly, reaching conflu-

ency in about 1 week.

Flow cytometry

According to flow cytometry results, most of thecells isolated from canine dental pulp tended toexpress CD90 and CD44 surface antigens. Otherstudied antigens, such as CD146, SSEA-4, and anti-macrophage, were expressed in very low percent-ages in the studied cells (Figure 2c through g).

Multilineage differentiation

Based on our regular observations of osteogeniccultures, some nodule-like structures appeared afew days after culture initiation and increased innumber over time. The nodules were the foci ofosteogenesis and tended to stain heavily withalizarin red, which specifically stains mineralizedmatrix (Figure 3a). The RT-PCR analysis revealed thatbone-related genes, including osteopontin andColIA1, were expressed in these cultures (Figure3b). In the adipogenic cultures, a few cells wereobserved that developed lipid droplets in theircytoplasm. These lipid droplets stained red with OilRed O stain (Figure 3c). Furthermore, based on theRT-PCR results, adipose related-genes, such aslipoprotein lipase and peroxisome proliferators,activated receptor-gamma expressed in the differ-entiated cells (Figure 3d). Positive toluidine bluestaining of the sections prepared from chondro-genic pellets demonstrated the production ofmetachromatic matrix at the chondrogenic cultures(Figure 3e). Chondrogenesis by dental pulp cellswas further analyzed and confirmed by RT-PCR.According to these analyses, cartilage-specificgenes, including aggrecan and collagen II, wereexpressed in the chondrogenic cultures (Figure 3f).

Scaffolds

Observation of the sections prepared from decalci-fied Bio-Oss loaded with DPSCs indicated thatDPSCs were successfully loaded into the biomaterialinternal pores (Figure 4). According to our results,about 60% of the loaded cells were successfullytrapped within the scaffold pore systems.

Clinical evaluation

Suppuration, infection, gingival recession, and rootexposure were not observed. There was nodifference between the test and control groupsduring the 8-week healing period. Initial inflamma-tion was comparable in both groups.

FIGURE 4. Photomicrograph of section prepared fromdecalcified Bio-Oss loaded with dental pulp stem cells(DPSCs). The inset indicates the rectangular area at a highermagnification. Bio-Oss internal pores loaded with DPSCs. Bar¼ 250 lm; original magnification, 310.

Journal of Oral Implantology 439

Khorsand et al

Histologic evaluation

In most histologic sections, Bio-Oss particles were

found in both groups and observed to be

surrounded by woven bone. Nevertheless, in 2

specimens from the control group a connective

tissue capsule was seen around the bone graft

particle (Figure 5a and b). The overall amount of

residual particles appeared to be similar in both

groups. Two specimens from the control group had

relatively more residual particles. In all test groups

and most of the control groups, epithelium

migration stopped at the coronal portion of the

coronal notch, but in 2 control groups, down

growth of the epithelium was seen in the apical

area of the coronal notch (Figure 5c and d).

Formation of new cementum, PDL, and bone wereobserved in all test specimens. In the controls, newbone was observed in all groups but the formationof cementum was seen in 9 defects and PDL wasseen in 8 defects (Figure 5e and f). Regeneratedcementum in the test group was thicker than that inthe control and covered a larger surface of the root.However, in the control groups, the cementumtended to be smaller, separated, and scattered.According to our findings, dentinoid (dentin-liketissue) formed in only one test group.

Histomorphometric evaluation

There was no significant difference in boneformation between the test and control groups.The amount of bone formation in these groups was

FIGURE 5. Histologic section from the repair tissue. (a) In some sections from the control, a connective tissue capsule (c) wasseen around the bone graft particle (b). Bar¼ 50 lm; original magnification, 350. (b) Test group: In most sections, Bio-Ossparticles were surrounded by woven bone (w). Bar ¼ 50 lm; original magnification, 350. (c) Test group: epitheliummigration stopped at the coronal portion of the coronal notch. Bar¼100 lm; original magnification, 310. (d) Control group:Down growth of epithelium was seen in the apical portion of the coronal notch. Bar¼ 100 lm; original magnification, 310.(e) A sample of control group containing granulation tissue (g) without any formation of bone, cementum, and periodontalligament (PDL). Bar¼100 lm; original magnification, 310. (f) A sample of test group showing perpendicular fibers of PDL (p)that formed between bone (b) and cementum (c). Bar ¼ 100 lm; original magnification, 325.

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DPSCs in Regeneration of Periodontal Tissue

3.60 6 1.06 mm in the test groups and 3.10 6 0.82mm in the control groups (Figure 6). New cemen-tum formation in the test groups was 3.83 6 1.32mm compared to 2.42 6 1.40 mm in the controlgroups (Figure 6), which was statistically significant(P , .05). Regarding PDL formation, the mean valuewas 3.30 6 1.12 mm for the test groups and 1.77 6

1.27 mm for the control groups (P , .05; Figure 6).

DISCUSSION

At present, the clinical results of periodontitistreatments are unsatisfactory. For this reason, newtherapies such as stem cell therapy have gainedconsiderable attention. DPSCs are adult stem cellsthat have several advantages over other types ofstem cells, including ease of accessibility andnoninvasive collection with low morbidity. Twogroups of studies have examined the capability ofDPSC for forming hard tissue. One group believesDPSCs, like bone marrow stem cells, differentiateinto osteoblasts and can produce bone.2,4,11 Theother group has shown that DPSCs differentiate intoodontoblasts and are able to generate dentin-liketissue.6,21–23 To date, no investigations have evalu-ated the potential of DPSCs in regeneration ofdefects in periodontal tissues that consist ofalveolar bone, cementum, and PDL. In the presentstudy, however, this was investigated. According toour findings, implantation of DPSCs combined withBio-Oss granules resulted in significant reconstruc-tion of cementum and PDL in addition to regener-ation of alveolar bone.

In the present study, DPSCs were isolated frommaxillary premolar teeth and characterized beforetransplantation in terms of some surface antigens.We used CD146 and anti-macrophage marker toexclude the endothelial origin and macrophagenature of the cells, respectively. SSEA-4 is anembryonic stem cell marker used to examinepluripotency of the isolated cells, and CD90 andCD44 are mesenchymal stem cell (MSC) markersused to ensure that the cells were from the MSCpopulation. The isolated cells possessed a tripotentdifferentiation potential capable of producing bone,cartilage, and adipose cells. Moreover, they grewadherent throughout the culture period. Thesecharacteristics were in agreement with propertiesproposed for MSCs by the Tissue Stem CellCommittee of the International Society for Cell

Therapy.24 Therefore, the isolated cells in this studycan be considered an MSC-like population with adental pulp origin.

According to our observations, woven boneformed at the implantation site. This differed fromformer studies in which lamellar bone formationwas reported after transplantation of DPSCs com-bined with collagen in rat calvaria.11 This differencemight be due to the difference in animal (dogversus rat) and biomaterial (Bio-Oss versus collagen)types that were used in the two studies. Theosteogenic effects of the DPSCs-collagen biocom-plex have also been mentioned by d’Aquino et al2

in a human mandible defect. According to theirfindings, the samples were made up of well-organized and well-vascularized bone with alamellar architecture that surrounded the haversianchannels.

In the area of cementogenesis and PDLformation, the present study is the first to evaluatethe potential of DPSCs. However, regeneration ofcementum and PDL with other dental stem cells,particularly PDLSCs, has already been investigated.Akizuki et al4 investigated periodontal regenerationby use of PDLSCs in defects surgically created onthe buccal surface of canine molars. In this studythey created a dehiscence defect compared to the3-walled defects of the current study, which aremore similar to periodontal defects. Liu et al3

explored the potential of PDLSCs to treat peri-odontal defects in a porcine model of periodontitis.They showed that PDLSCs are capable of regener-ating periodontal tissues. Liu et al3 quantified boneformation but not cementum and PDL formation,whereas in the present study, we quantified alltissue components that make up the periodon-tium. A study by Dogan et al5 is another exampleof the use of PDL-derived stem cells in theregeneration of periodontal tissue in caninemodels. This was a pilot study where a furcationdefect was created in just one dog; however, in ourstudy 10 dogs were used to assess DPSCs in 3-walled defects.

All the aforementioned cell-seeding studies maybe useful and promising; however, numerousquestions have been posed. It is recommendedthat researchers perform other studies that useanother type of control group, such as autografts orother biomaterials that can be loaded with thegrowth factors. Furthermore, at this time there are

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numerous additional ways to examine regeneration

and prove that the tissues produced are of the

desired type, such as immunohistochemistry to

identify tissue-specific antigens, in situ hybridization

to demonstrate tissue-specific gene expression,

micro-computed tomography to demonstrate the

regeneration of mineralized tissue, and bone

density measurements to analyze calcification of

regenerated tissues. Without a broader range of

experimental analyses the results are limited to very

descriptive assessments of what the new tissues

look like, which is not sufficient to demonstrate

what the tissues actually are.

CONCLUSION

This investigation is the first study showing that

DPSCs are capable of promoting periodontal

regeneration. Along with a Bio-Oss scaffold, DPSCs

would be an excellent biocomplex. In fact, although

DPSCs seem to be a promising therapy in the

treatment of periodontal diseases, additional re-

search is needed in this area.

ABBREVIATIONS

DMEM: Dulbecco’s modified eagle medium

DPSC: dental pulp stem cell

FBS: fetal bovine serum

MSC: mesenchymal stem cell

PBS: phosphate buffered saline

PDL: periodontal ligament

PDLSC: periodontal ligament stem cell

RT-PCR: reverse transcriptase-polymerase chain

reaction

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