Basic Research—Biology

Transplantation of Dental Pulp Stem Cells and Platelet-richPlasma for Pulp RegenerationXiaofei Zhu, DDS, PhD,*† Chengfei Zhang, DDS, PhD,† George T.-J. Huang, DDS, MSD, DSc,‡

Gary S.P. Cheung, DDS, PhD,†Waruna Lakmal Dissanayaka, BDS,

†and Wenhao Zhu, DMS

§

Abstract

Introduction: The loss of dental pulp may weakenteeth, rendering them susceptible to reinfection, frac-ture, and subsequent tooth loss. Therefore, regenerationof pulp is considered an ideal treatment to preserveteeth. The aim of this study was to explore the capacityof dental pulp stem cells (DPSCs) and platelet-richplasma (PRP) to regenerate dental pulp in canine maturepermanent teeth. Methods: Pulpectomy with apicalforamen enlarged to a #80 file was performed in 16upper premolars of 4 beagle dogs. Four experimentalgroups were randomly established: (1) the blood clotgroup, (2) the autologous DPSCs group, (3) the PRPgroup, and (4) the DP + PRP group (a mixture of DPSCsand PRP). Four lower premolars without any furthertreatment after pulpectomy were used as the controlgroup. All teeth were sealed with mineral trioxide aggre-gate and composite. Twelve weeks after transplantation,the teeth were subjected to radiographic and histologicexamination. Results: Twenty-four of 32 experimentalroot canals gained newly formed tissues. All canalswith an introduction of a blood clot showed histologicevidence of vital tissue formation. Cementum-like andperiodontal ligament–like tissues along the internalroot canal walls were typical structures in most cases.There is no significant difference between groups withor without autologous DPSC transplantation (exact chi-square test, P < .05). Conclusions: New vital tissuescan be regenerated in permanent canine teeth after pul-pectomy and enlargement of the apical foramen. Histo-logically, transplantation of DPSCs and/or PRP into rootcanals showed no enhancement in new tissue formationcompared with inducement of a blood clot into the rootcanals alone. (J Endod 2012;38:1604–1609)

Key WordsDental pulp stem cells, permanent teeth, platelet-richplasma, pulp regeneration, vital tissue growth

From the *Special Department, School and Hospital of Stomatolosity of Hong Kong, Hong Kong, China; ‡Department of Endodontics,Dentistry, School and Hospital of Stomatology, Peking University, B

Supported by International scientific and technological cooperatio2009DFA32950) and GRF grants from the Research Grants Council

Address requests for reprints to Dr Chengfei Zhang, Compreheaddress: zhangcf@hku.hk0099-2399/$ - see front matter

Copyright ª 2012 American Association of Endodontists.http://dx.doi.org/10.1016/j.joen.2012.09.001

1604 Zhu et al.

The emergence of regenerative medicine has prompted the interest in endodontics totest the possibility of dental pulp and dentin regeneration. It was considered that

pulp/dentin regeneration is preferable to pulpectomy/gutta-percha obturation fromthe perspective of preserving tooth longevity (1, 2). Two approaches have beenundertaken to test pulp/dentin regeneration. One is the revitalization approach(revascularization) to treat endodontically involved immature permanent teeth.Multiple clinical case reports have shown a certain level of success after revitalizationtreatment in terms of the absence of clinical symptoms, the healing of periapicallesions, and the increase of root thickness and length based on radiographicevidence (3, 4). Animal studies have provided both radiographic and histologicevidence of tissue generated in canals after the revitalization of immature permanentteeth with experimentally induced apical periodontitis (5, 6). It was found that therewas no regeneration of pulp/dentin in the canals; instead, they were filled withperiodontal tissues including cementum, periodontal ligament, and bone (6).

The other approach to regenerate pulp/dentin is to introduce exogenous stemcells into the canals. In an ectopic regeneration study model, human dental pulp/dentin-like tissues were formed in the emptied canals of human tooth fragments usinga heterogeneous population of stem cells from apical papilla (SCAPs) or dental pulpstem cells (DPSCs) (7). Although heterologous DPSCs showed a relatively low immu-nologic response in animal studies, autologous DPSC transplantation is obviously lessrisky. Complete pulp regeneration also has been shown orthotopically in a large animalmodel by transplanting pulp CD105+ cells with stromal cell–derived factor-1 intomature teeth with the apex enlarged only �0.7 mm (8).

The immature tooth has an open apex, large canal, and a short root. The new tissuecan easily grow into the root canal space, reaching the coronal pulp chamber (9). Apermanent tooth with a mature apex may have a limited blood supply to allow tissueingrowth into the canal (10). Reimplantation of avulsed teeth with an apical openingof �1.0 mm in diameter showed a greater likelihood of revascularization (11, 12),which indicates that regeneration of dental pulp in teeth with mature apices might beachievable by enlarging the apex to 1–2 mm in diameter.

A recent clinical case report showed that revitalization of an endodonticallycompromised immature tooth by using platelet-rich plasma (PRP) may provide a desir-able outcome (13). PRP contains many growth factors including platelet-derivedgrowth factor, transforming growth factor b, and insulin-like growth factor (14) andmay be a good supplement for cell-based pulp/dentin regeneration. PRPmay be derivedfrom patient’s own blood, is easy to prepare, and is capable of forming a 3-dimensionalfibrin matrix (that acts as a scaffold) (15–17). An in vitro study showed that PRP can

gy, Peking University, Beijing, China; †Comprehensive Dental Care, Faculty of Dentistry, The Univer-Boston University School of Dental Medicine, Boston, Massachusetts; and §Department of Generaleijing, China.n projects from The Ministry of Science and Technology of the People’s Republic of China (grant no.of Hong Kong (grant no. HKU 785010M).nsive Dental Care, Faculty of Dentistry, The University of Hong Kong, Hong Kong, China. E-mail

JOE — Volume 38, Number 12, December 2012

Basic Research—Biology

enhance the proliferation and differentiation of human DPSCs (18). Inthis study, we aimed to investigate the potential synergistic effects ofautologous DPSCs and PRP for de novo dental pulp regeneration inthe root canals after pulpectomy in a dog study model.

Materials and MethodsAnimals

Four beagles approximately 1 year old were obtained from theExperimental Animal Center of the Peking University Health ScienceCenter, Beijing, China. Animal care and handling followed the guide-lines of the Institutional Authority for Laboratory Animal Care, PekingUniversity. This study was reviewed and approved by the Health ScienceCenter, Peking University.

Isolation, Culturing, and Identificationof Canine Dental Pulp Stem Cells

Autologous canine DPSCs (cDPSCs) were isolated fromfreshly extracted incisors (n = 4) of each beagle as previouslydescribed (19). Briefly, tooth surfaces of the freshly extractedteeth were cleaned and cut at the cementoenamel junction usinga sterile fissure bur to reveal the pulp chamber. After separatingthe pulp tissue gently from the crown and root, it was digestedin a 3-mg/mL collagenase type I (GIBCO-Invitrogen, Carlsbad,CA) and 4- mg/mL dispase (GIBCO-Invitrogen) solution for 1hour at 37�C. Then, the cells were passed through a 70-mmstrainer (BD Falcon, Franklin Lakes, NJ) to obtain single-cellsuspensions. These cells were seeded in 75-cm2 culture flaskscontaining a–minimum essential medium supplemented with15% fetal bovine serum, L-ascorbic acid-2- phosphate, 100 U/mL penicillin-G, 100 mg/mL streptomycin, and 0.25 mg/mL Fun-gizone (Gemini Bio-Products, Woodland, CA) and cultured under5% CO2 at 37�C. Medium was replaced every 3 days, and cellswere subcultured at 70% confluence. The expression of mesen-chymal stem cell markers was analyzed on a fluorescenceactivated cell sorter caliber flow cytometer. cDPSCs were STRO-1(+), CD146(+), CD73(�), CD105(�), and CD45(�) as previ-ously described (19). Multilineage differentiation potential intoosteo/odontogenic, adipogenic, and neurogenic lineages wasconfirmed (data not shown). cDPSCs at passages 3 to 4 wereharvested, centrifuged, and washed 3 times with physiologicalsaline before transplanting into the root canals.

Preparation of Activated PRPPRP was procured using a previously described method (20) with

minor modification. Approximately 20 mL blood was drawn from eachdog into a centrifuge tube containing 3 mL citrate solution. Collectedblood was centrifuged for 10 minutes at 200g to obtain PRP withouterythrocytes and leukocytes. A second centrifugation was performedfor 15 minutes at 360g. PRP was taken, and the platelet-poor plasmawas removed (18). Platelets in whole blood and PRP were countedwith an automatic hematology analyzer to make sure that plateletconcentration was more than 1,200� 109/L. Bovine thrombin (Sigma,St Louis, MO) was combined with 10% calcium chloride in a proportionof 1,000 U thrombin/1 mL CaCl2. The release of platelet products intothe supernatant was induced by adding 200 IU activated thrombin intoeach PRP sample.

In Vivo Transplantation StudyRoot Canal Preparation. The protocol of whole pulp removalfrom permanent premolars was established in beagle dogs as

JOE — Volume 38, Number 12, December 2012

described previously (8). Briefly, under general anesthesia (ie,induction by pentothal 13.5 mg/kg intravenously and intubationand maintenance with isoflurane) supplemented with local anes-thesia (ie, 4% articaine with 1:100,000 epinephrine), the pulpwas mechanically exposed with a #2 round carbide bur ina high-speed handpiece and taken out by a barbed broach. A sterile#15 K-file (Dentsply Maillefer, Johnson City, TN) was used to nego-tiate to the apex as determined by preoperative radiographs. Then,the root canals were prepared followed by the enlargement of theapical foramen to 0.8 mm in diameter using a #80 K-file with5.25% sodium hypochloride (NaOCl) and 17% EDTA irrigation.Each prepared canal received a final rinse with 5 mL physiologicalsaline and was dried with sterile paper points before the subsequentprocedures described later.

Experimental and Control Groups. Sixteen double-rootedupper premolar teeth (second and third upper premolar) in 4beagles were divided into 4 experimental groups, and 4 lowerpremolars (1 from each dog) were used as the control group.Two canals in the same tooth were used for the same experimentalprocedures. The sample size (n) is referred to as the number ofroot canals.

For the blood clot (BC) group (n = 8 root canals),bleeding into canals was evoked by overinstrumentation intoapical tissues with a #80 K-file allowing the formation of a bloodclot. For the cDPSC (DP) group (n = 8 root canals), autologouscDPSCs (1 � 106 cells) were suspended in 20 mL vein bloodand syringed into each root canal, whereas for the PRP group(n = 8 root canals), 20 mL PRP was injected into each rootcanal immediately after being activated. Finally, for the cDPSCsplus PRP (DP + PRP) group (n = 8 root canals), cDPSCs(1 � 106 cells) and 20 mL activated PRP were mixed and in-jected into each root canal. A patency check to the apex was per-formed with a sterile #15 K-file after transplantation in DP, PRP,and DP + PRP groups to make sure the root canal and the mate-rial transplanted connected with periapical tissue directly. For thecontrol group, 4 lower premolars (1 from each dog) receivedthe endodontic procedure, and the canals were left empty. Thecoronal portions of the root canals were double sealed by whitemineral trioxide aggregate (MTA) (Dentsply Tulsa Dental, John-son City, TN) and composite (P60; 3M Dental Products,St Paul, MN).

Preoperative and 3-month postoperative radiographs were taken.The presence or absence of apical radiolucency was evaluated by 2independent examiners. The animals were sacrificed under generalanesthesia (pentobarbital; Butler Company, Columbus, OH) at 30mg/kg intravenously. The jaws with the involved teeth were resectedand fixed for histologic processing.

Histologic AnalysisAfter fixation in 4% paraformaldehyde for 24 hours and decal-

cification in 10% EDTA for 6 months, the specimens wereembedded in paraffin wax, sectioned longitudinally along thelong axis of the teeth, and stained with hematoxylin-eosin. Eachindividual root was analyzed as an independent sample unit histo-logically under a light microscope for the presence or absence ofregenerated pulp tissue and the structure of the generated vitaltissues.

Data AnalysisThe categoric data (whether regenerated tissue was present or

not) were analyzed with exact chi-square tests, with the level of

Dental Pulp Regeneration with PRP and DPSCs 1605

Figure 1. (A) A preoperative radiograph. (B) Twelve weeks after the regenerative endodontic procedure, periapical radiolucency was detected from the secondupper premolar (circle). (C) Histologic examination showed the inflammatory cell infiltration and bone loss around the apex (arrow).

Figure 2. Twenty-four of 32 experimental roots got newly formed vitaltissues. Two roots cracked during the histologic sectioning procedure andwere excluded from the study. Periodontitis was detected in 6 roots. No signif-icant difference on the rate of vital tissue formation in the canals can be foundbetween groups (exact chi-square test, P < .05).

Basic Research—Biology

significance set at P < .05, to determine if there were any significantdifferences between the experimental groups with respect to the histo-logic criteria.

ResultsClinical and Radiologic Findings

All 16 experimental teeth showed no signs of mobility swellingor sinus tracts. All fillings were intact. Periapical radiolucency wasdetected in 8 teeth (2 teeth in the DP group, 1 tooth from the PRPand DP + PRP groups, and 4 teeth in the control group) and wasconfirmed as periapical periodontitis by histologic examination(Fig. 1A–C).

Histologic Findings of Vital Tissues in the Canal SpaceNewly formed vital tissues were found in the canal space in 24

of 32 roots in the experimental groups. The data in Figure 2present the number of root canals with newly generated tissuein different groups. Two roots (from the DP and DP + PRPgroups) were excluded because of cracking during the histologicslice procedure. No tissue or only some inflammatory cells inapical portion were found in 6 roots (from 4 teeth), and thesesamples were sorted as necrotic (Fig. 2). Periapical periodontitiswas also detected in these 6 roots. In the BC group, new vitaltissue was present in all 8 roots. The absence of new vital tissuewas found in 3 root canals in the DP group. The PRP and DP +PRP groups had 2 and 1 canals without new vital tissue, respec-tively. All 6 canals that had no new vital tissue inside presentedinflammatory cell infiltration in the periapical area, which corre-lates with radiologic findings. The categoric data on the formationof vital tissues in the canal space from each group were 8 out of 8teeth in the BC group, 4 out of 7 teeth in the DP group, 6 out of 8teeth in the PRP group, and 6 out of 7 teeth in the DP + PRPgroup. There was no significant difference between groups (exactchi-square test, P < .05). Hard-tissue deposition along internalroot canal walls was detected in 23 of 24 that had vital tissuesgenerated. Twenty of these 24 root canals showed a bone-likestructure inside the newly formed tissues.

Histologic Characteristics of Vital Tissuesin the Root Canal Space

Eighteen cases of the newly generated tissues extended to thesurface of the MTA and 6 cases to the midlevel of the root canals(Figs. 2 and 3A and B). In the coronal part in which tissues were in

1606 Zhu et al.

direct contact with MTA or in close promixity, osteoid hard tissuewas observed. Some particles embedded in the newly formedosteoid tissues may have been the MTA; cells in the middleportion were stellate-like (processes radiating from the cell bodygiving them a spindle or stellate shape), whereas those in the apicalthird were more immature with larger and heavily stained nuclei(Fig. 4B–D). Among all specimens with vital tissues in the canals,no dentin-like tissue was detected, whereas cementum-like tissueand periodontal ligament–like tissue were observed along thedentinal wall (Fig. 4E–G).

Cementum-like Tissue Depositionalong Internal Root Canal Walls

An aavascular mineralized tissue (cementum-like tissue)covering along the internal root canal walls was observed in nearlyall root canals with vital tissues (except 1 canal from the PRP group).The cementum-like tissue showed no difference in structure amongthe 4 groups. Continuation of the cementum from the outer rootsurface into the inner canal surface was observed in most cases(Fig. 3C and D). The thickness of the cementum-like tissue decreasedfrom the apical to the coronal area (Fig. 3E and F). Cementocyte-likecells were found embedded in this tissue (Fig. 3J), and cementoblast-like cells were seen on the surface of the cementum-like tissue(Fig. 3G and I). Immediately adjacent to the cementoblast-like layerin the canal, a layer of soft tissue resembling the periodontal ligament

JOE — Volume 38, Number 12, December 2012

Figure 3. (A) A full-length regenerated case (from the DP+PRP group); the tissue has grown above the cervical level up to the pulp horns. (B) A half-length regeneratedcase (from the DP + PRP group); the tissue growth has stopped at the midlevel of the root. Some inflammatory cells can be seen above the tissue. (C and D) Ingrowth ofcementum-like tissue from the outer root surface into the inner canal surface. (E and F) The thickness of cementum-like tissue has reduced from the apical to the coronaldirection. Bone-like structure was shown in the section. (G) Cementum-like tissue deposition along the internal root canal walls. (H) A magnified view shows the combi-nation of dentin (D), cementum-like tissue (C), and periodontal ligament–like tissue (P). PS, pulp space. (I) Cementoblast-like cells lie along cementum-like tissue, andsome fibers from the PDL-like tissue were inserted into the cementum-like tissue, resembling Sharpey’s fibers. (J) An embedded cementocyte-like cell (arrow).

Basic Research—Biology

JOE — Volume 38, Number 12, December 2012 Dental Pulp Regeneration with PRP and DPSCs 1607

Basic Research—Biology

was observed in 18 samples (Fig. 3H). Sharpey’s fibers were foundinserting from the PDL-like tissue into the cementum-like tissue(Figs. 3I and 4G).

Bone-like Tissue Formation in the NewlyRegenerated Tissue

There was histologic evidence of islets of bone-like matrixwith strong eosinophilic staining in hematoxylin-eosin–stainedsections. Some cells were also seen embedded in the matrix.Twenty experimental roots showed bone-like tissue in the rootcanal space (2 roots from the PRP group and 2 roots from theDP + PRP group showed no evidence on bone-like structure).These structures were commonly present in the coronal part ofthe root canal, especially in the zone close to or directly connectedwith MTA (Figs. 3E and F and 4A and D). In some cases, bone-liketissue resembled cementum-like tissue without apparent bonemarrow space.

DiscussionSeveral revitalization studies in humans and animals have shown

that after disinfection and evoking blood into root canals, the rootthickening and lengthening could be achieved (4, 5, 13, 21).However, it was shown that the newly grown tissues into the rootcanal space have little similarity to normal pulp tissue but withmore resemblance to cementum, periodontal ligament, or bone.The cause of this outcome is possibly related to the lack of stemcells derived from remaining vital pulp and apical papilla, whichare destroyed by severe endodontic infection. Stem cells responsiblefor newly regenerated tissues might be derived from several othersources, including systemic blood, local tissue such as bone, andthe periodontal ligament (22). Furthermore, whether these newlyformed tissues can function like normal pulp and stabilize the toothwithout giving rise to further infection or canal obliteration still

Figure 4. (A) A full-length regenerated case (from the PRP group). (B) The apicshowed stellate-like appearance resembling the normal pulp cells. (D) Cells at the c(C) and periodontal ligament–like (P) structures along the dentinal wall (D).

1608 Zhu et al.

remains unclear (23). Therefore, more research is needed beforethe regenerative procedures can be routinely performed with a predict-able long-term prognosis.

The triad of cells, growth factors, and scaffolds is necessary forappropriate tissue regeneration. It has been hypothesized that afterdisinfection stem cells from the remaining vital pulp or apical papillamay reconstitute the lost structure of pulp/dentin complex (6).However, endogenous DPSCs and SCAP might not be able to survivefrom severe endodontic infection. In the present study, we attemptedto deliver DPSCs into the canal space filled with blood or DPSCsmixed with PRP and compared these approaches with a bloodclot alone.

In contrast to our expectation, the histologic examination did notdisplay any difference between the BC and DPSC transplantation groups.One possible reason may be that fractionation of stem cells and propergrowth factors is a more favorable approach for dental pulp regenera-tion. According to studies by Iohara et al (8), they also found that trans-plantation of unfractionated total pulp cells into root canal showed lesstissue formation followed by evidence of mineralization on day 90compared with transplantation of CD105+ pulp cells and stromalcell–derived factor-1. Although DPSCs are the most direct cell sourcein dental pulp regeneration, a number of other cell sources includingSCAPs, periodontal ligament stem cells, and bonemarrowmesenchymalstem cells may also contribute to dental pulp regeneration (24). Thismay be another reason why the transplantation of autologous DPSCsalone did not help dental pulp regeneration in the present study. Furtherstudies are needed to identify the cell sources of the tissues formed inthe canal space (ie, from periapical tissues or from the transplantedDPSCs).

Growth factors and a suitable scaffold are also essential consid-erations in tissue regeneration. PRP contains several growth factorsincluding transforming growth factor beta 1, platelet-derived growthfactor, fibroblast growth factor, vascular endothelial growth factor,and epidermal growth factor (25, 26) that support cell growth,

al cells were heavily stained with large nuclei. (C) Cells in the middle portionoronal part differentiated into irregular bone-like tissue. (E–G) Cementum-like

JOE — Volume 38, Number 12, December 2012

Basic Research—Biology

differentiation, and migration of DPSCs (27, 28). PRP is also capableof forming a 3-dimensional fibrin matrix, which acts as a scaffold (15,16). Therefore, PRP has been suggested as a potential scaffold forregenerative endodontic procedure (15); 1 clinical case reportappears to support this idea (13). Although PRP has been widelyused in treatments of bone defect in periodontitis (29), some animaland clinical studies found that PRP does not appear to enhance boneregeneration (30, 31). Limited information is available on the use ofPRP for dental pulp regeneration. In the present study, we found thatPRP alone or the combination of PRP and DPSCs did not enhance theregeneration of pulp-like tissues. It is possible that the collagen scaf-fold used by Iohara et al (8) to carry DPSCs into the canals mayprovide a better condition for pulp regeneration compared withPRP used in the present study. The in vitro study showed thatalthough PRP can enhance mineralization differentiation of DPSCs(18), it is not clear whether PRP enhances dentinogenesis (ie, PRPmay not promote pulp-dentin regeneration).

Newly formed tissues in the canals could extend to the surface ofMTA in some cases or occupy half of the canal space after 3 months. Thegrowth of the tissue into the canal seemed not limited by the bloodsupply with an apical opening of 0.8 mm in diameter. When tissues en-gineered in the laboratory are implanted into the human body, only cellswithin 100–200 mm from the nearest capillary can attain sufficientdiffusion of nutrients to survive. Thus, it was suggested that a voluminoustissue be prevascularized for achieving immediate and sufficient bloodsupply after implantation (32). It would be reasonable to infer that oncethe newly generated vasculature inside canals is connected with periap-ical tissues, the middle and coronal portion of pulp-like tissue couldalso be incrementally formed from the apical portion after 3 months.

The newly formed tissue showed no obvious difference among the4 experimental groups in our study. Bone-like structures were found inmost of the cases, especially in the coronal portion or close to the MTA.MTA is a good material for pulp capping and apexification, which caninduce dental pulp cell differentiation and the secretion of mineralizedtissue (33). Cells in new vital tissues in the apical canal were moreimmature with larger and deeply stained nuclei. These cells pertainmore potential for multilineage differentiation. Cementum-like tissueand periodontal ligament-like tissue were seen along the internalroot canal walls. In some cases, cementum-like tissue inside the rootcanal was connected with root surface cementum. The source ofstem cells responsible for bone-like, periodontal ligament–like, andcementum-like tissues is not clear, possibly from the periapical tissues.

AcknowledgmentsThe authors deny any conflicts of interest related to this study.

References1. Nakashima M, Akamine A. The application of tissue engineering to regeneration of

pulp and dentin in endodontics. J Endod 2005;31:711–8.2. Huang GT. Dental pulp and dentin tissue engineering and regeneration: advance-

ment and challenge. Front Biosci (Elite Ed) 2011;3:788–800.3. Ding RY, Cheung GS, Chen J, et al. Pulp revascularization of immature teeth with

apical periodontitis: a clinical study. J Endod 2009;35:745–9.4. Bose R, Nummikoski P, Hargreaves K. A retrospective evaluation of radiographic

outcomes in immature teeth with necrotic root canal systems treated with regener-ative endodontic procedures. J Endod 2009;35:1343–9.

5. Thibodeau B, Teixeira F, Yamauchi M, et al. Pulp revascularization of immature dogteeth with apical periodontitis. J Endod 2007;33:680–9.

JOE — Volume 38, Number 12, December 2012

6. Wang X, Thibodeau B, Trope M, et al. Histologic characterization of regeneratedtissues in canal space after the revitalization/revascularization procedure of imma-ture dog teeth with apical periodontitis. J Endod 2010;36:56–63.

7. Huang GT, Yamaza T, Shea LD, et al. Stem/progenitor cell-mediated de novo regen-eration of dental pulp with newly deposited continuous layer of dentin in an in vivomodel. Tissue Eng Part A 2010;16:605–15.

8. Iohara K, Imabayashi K, Ishizaka R, et al. Complete pulp regeneration after pulpec-tomy by transplantation of CD105+ stem cells with stromal cell-derived factor-1.Tissue Eng Part A 2011;17:1911–20.

9. Trope M. Regenerative potential of dental pulp. J Endod 2008;34:S13–7.10. Huang GT, Sonoyama W, Liu Y, et al. The hidden treasure in apical papilla: the

potential role in pulp/dentin regeneration and bioroot engineering. J Endod2008;34:645–51.

11. Kling M, Cvek M, Mejare I. Rate and predictability of pulp revascularization in ther-apeutically reimplanted permanent incisors. Endod Dent Traumatol 1986;2:83–9.

12. Murray PE, Garcia-Godoy F, Hargreaves KM. Regenerative endodontics: a review ofcurrent status and a call for action. J Endod 2007;33:377–90.

13. Torabinejad M, Turman M. Revitalization of tooth with necrotic pulp and open apexby using platelet-rich plasma: a case report. J Endod 2011;37:265–8.

14. Slavkin HC, Bartold PM. Challenges and potential in tissue engineering. Periodontol2000 2006;41:9–15.

15. Hargreaves KM, Giesler T, Henry M, Wang Y. Regeneration potential of the youngpermanent tooth: what does the future hold? J Endod 2008;34:S51–6.

16. Anitua E, Sanchez M, Nurden AT, et al. New insights into and novel applications forplatelet-rich fibrin therapies. Trends Biotechnol 2006;24:227–34.

17. Ogino Y, Ayukawa Y, Kukita T, Koyano K. The contribution of platelet-derived growthfactor, transforming growth factor-beta1, and insulin-like growth factor-I in platelet-rich plasma to the proliferation of osteoblast-like cells. Oral Surg Oral Med OralPathol Oral Radiol Endod 2006;101:724–9.

18. Lee UL, Jeon SH, Park JY, Choung PH. Effect of platelet-rich plasma on dental stemcells derived from human impacted third molars. Regen Med 2011;6:67–79.

19. Dissanayaka WL, Zhu X, Zhang C, Jin L. Characterization of dental pulp stem cellsisolated from canine premolars. J Endod 2011;37:1074–80.

20. Nikolidakis D, Jansen JA. The biology of platelet-rich plasma and its application inoral surgery: literature review. Tissue Eng Part B Rev 2008;14:249–58.

21. Yamauchi N, Yamauchi S, Nagaoka H, et al. Tissue engineering strategies for imma-ture teeth with apical periodontitis. J Endod 2011;37:390–7.

22. Lovelace TW, Henry MA, Hargreaves KM, Diogenes A. Evaluation of the delivery ofmesenchymal stem cells into the root canal space of necrotic immature teeth afterclinical regenerative endodontic procedure. J Endod 2011;37:133–8.

23. Andreasen JO, Bakland LK. Pulp regeneration after non-infected and infectednecrosis, what type of tissue do we want? A review. Dent Traumatol 2012;28:13–8.

24. Andreasen JO. Pulp and periodontal tissue repair—regeneration or tissue meta-plasia after dental trauma. A review. Dent Traumatol 2012;28:19–24.

25. Marx RE, Carlson ER, Eichstaedt RM, et al. Platelet-rich plasma: growth factorenhancement for bone grafts. Oral Surg Oral Med Oral Pathol Oral Radiol Endod1998;85:638–46.

26. Schilephake H. Bone growth factors in maxillofacial skeletal reconstruction. Int JOral Maxillofac Surg 2002;31:469–84.

27. Howard C, Murray PE, Namerow KN. Dental pulp stem cell migration. J Endod 2010;36:1963–6.

28. Osathanon T, Nowwarote N, Pavasant P. Basic fibroblast growth factor inhibitsmineralization but induces neuronal differentiation by human dental pulp stem cellsthrough a FGFR and PLCgamma signaling pathway. J Cell Biochem 2011;112:1807–16.

29. Pradeep AR, Rao NS, Agarwal E, Bajaj P. Comparative evaluation of autologousplatelet-rich fibrin and platelet-rich plasma in the treatment of three-wall intrabonydefects in chronic periodontitis: a randomized controlled clinical trial. J Periodontol2012 [Epub ahead of print].

30. Ranly DM, Lohmann CH, Andreacchio D, et al. Platelet-rich plasma inhibits demin-eralized bone matrix-induced bone formation in nude mice. J Bone Joint Surg Am2007;89:139–47.

31. Raghoebar GM, Schortinghuis J, Liem RS, et al. Does platelet-rich plasma promoteremodeling of autologous bone grafts used for augmentation of the maxillary sinusfloor? Clin Oral Implants Res 2005;16:349–56.

32. Hendrickx B, Vranckx JJ, Luttun A. Cell-based vascularization strategies for skintissue engineering. Tissue Eng Part B Rev 2011;17:13–24.

33. Paranjpe A, Zhang H, Johnson JD. Effects of mineral trioxide aggregate on humandental pulp cells after pulp-capping procedures. J Endod 2010;36:1042–7.

Dental Pulp Regeneration with PRP and DPSCs 1609