2008;139(4):457-465JADA Garcia-GodoyNamerow, Sergio Kuttler and Franklin Eric L. Gotlieb, Peter E. Murray, Kenneth N.Within Endodontically Treated Teeth
ImplantedTissue-Engineered Pulp Constructs An Ultrastructural Investigation of
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Background. The authors conducted an ultrastructural scanning elec-tron microscopic (SEM) investigation of tissue-engineered pulp constructsimplanted within endodontically treated teeth.Methods. Stem cells from human exfoliated deciduous teeth wereseeded on a synthetic open-cell D,D-L,L-polylactic acid scaffold with orwithout the addition of bone morphogenic protein-2 and transforminggrowth factor β1 to create pulp tissue constructs. The pulp constructswere implanted into 105 extracted human premolar teeth with a singleroot canal that had been cleaned and shaped by using rotary instrumen-tation in a crown-down manner to ISO size no. 35.Results. An ultrastructural examination of the SEM micrographs at ×2,000 magnification revealed cell adherence within all of the pulp con-structs, with little difference between the scaffold types or with the addi-tion of growth factors.Conclusions. These results support the proof-of-concept that it is pos-sible to implant tissue-engineered pulp constructs into teeth aftercleaning and shaping.Clinical Implications. Future regenerative endodontic treatmentmay involve the cleaning and shaping of root canals followed by theimplantation of vital dental pulp tissue constructs created in the laboratory.Key Words. Stem cells; root canal; endodontics; pulpitis; dental pulptissue constructs.JADA 2008;139(4):457-465.
The most valuable cells forregenerative dentistryare stem cells.1 A stemcell is commonly definedas a cell that has the
ability to divide continuously andproduce progeny cells that differen-tiate (develop) into various othertypes of cells or tissues.2 The dentalpulp contains a stem cell populationknown as pulp stem cells3,4 or, in thecase of immature teeth, stem cellsfrom human exfoliated deciduousteeth (SHED).5,6
To create a practical endodontictissue-engineering therapy, investi-gators must organize pulp stemcells into a three-dimensional struc-ture using tissue-engineering scaf-folds. Several studies of dental pulpcells seeded on 3-D scaffolds havebeen completed. Recently, Zhangand colleagues7 observed successfuldental pulp stem cell growth onspongeous collagen, porous ceramicand fibrous titanium mesh scaffoldsthat were implanted into nude mice.Hee and colleagues8 found that bio-ceramic calcium phosphate tissuescaffolds appear more optimal forbone regeneration in comparisonwith polymer scaffolds. However,biodegradable polymer scaffoldsmanufactured from the same bioma-terials used in surgical dissolvablesutures9,10 have an advantage inthat these biomaterials have beenproven to be biocompatibleaccording to screening assaysapproved by the U.S. Food and
A B S T R A C T
At the time this study was conducted, Dr. Gotlieb was a resident, Department of Endodontics, Collegeof Dental Medicine, Nova Southeastern University, Fort Lauderdale, Fla. He now is in private practice,Exeter Endodontics, Exeter, N.H.Dr. Murray is an associate professor, Department of Endodontics, College of Dental Medicine, NovaSoutheastern University, 3200 S. University Drive, Fort Lauderdale, Fla. 33328-2018, e-mail “[email protected]”. Address reprint requests to Dr. Murray.Dr. Namerow is an associate professor and chair, Department of Endodontics, College of Dental Medi-cine, Nova Southeastern University, Fort Lauderdale, Fla.Dr. Kuttler is an associate professor and assistant dean for postgraduate education, College of DentalMedicine, Nova Southeastern University, Fort Lauderdale, Fla.Dr. Garcia-Godoy is a professor and associate dean for research, College of Dental Medicine, NovaSoutheastern University, Fort Lauderdale, Fla.
An ultrastructural investigation of tissue-engineered pulp constructs implantedwithin endodontically treated teethEric L. Gotlieb, DDS; Peter E. Murray, PhD; Kenneth N. Namerow, DDS; Sergio Kuttler, DDS;Franklin Garcia-Godoy, DDS, MS
Several studies have suggested that polymerscaffolds hold the most promise for creatingreplacement tissues through tissue engineering.Human dental pulp and gingival fibroblastsadhere, proliferate and produce extracellularmatrix when seeded on polymer scaffolds invitro.12 Researchers also have used polymer scaf-folds to bioengineer tooth tissues from porcine13
and rodent tooth bud cells,14 but they have not yetseeded polymer scaffolds with human dental pulpstem cells or SHED to be investigated for dentalpulp tissue engineering.
The seeding of cells on tissue engineering scaf-folds is known as “creating a tissue construct.”15
To promote the formation of higher-ordered tissuestructures, tissue constructs are maintained incell culture in the presence of growth factors orbioactive molecules. Growth factors, especiallythose of the transforming growth factor β (TGFβ)family, are important in cellular signaling forodontoblast differentiation and stimulation ofdentin matrix secretion. These growth factors aresecreted by odontoblasts and are deposited withinthe dentin matrix, where they remain protectedin an active form through interaction with othercomponents of the dentin matrix.16 The additionof purified dentin protein fractions has stimulatedan increase in tertiary dentin matrix secretion,suggesting that TGFβ1 is involved in injury-signaling and tooth-healing reactions.17
Another important growth factor in toothdevelopment and regeneration is bone mor-phogenic proteins (BMPs).18 Recombinant humanBMP-2 stimulates differentiation of adult pulpstem cells into an odontoblastoid morphology inculture.19 Researchers have demonstrated thesimilar effects of TGFβ1-3 and BMP-7 in culturedtooth slices.20 Recombinant BMP-2, -4 and -7induce reparative dentin in vivo.21 These studies18-21
suggest that the addition of growth factors to theculture media may promote the formation ofdental pulp tissue constructs. However, it isunclear if BMP-2 or TFGβ1 is optimal, and it isnot known what effects these growth factors haveon tissue-engineered constructs.
Numerous studies have demonstrated thatcells can attach to and grow on dentin surfaces,while other studies have shown that cells canattach to tissue-engineering scaffolds.22 However,no previous studies, to our knowledge, have inves-tigated the potential of SHED to create dentalpulp constructs within human cleaned and
shaped root canals, which has been suggested asa possible future endodontic regenerative treatment.22
Therefore, the purpose of this study was toexamine how SHED attachment within a porouspolymer scaffold can be used to create a dentalpulp tissue construct. The variables to be testedincluded the longevity of the cell culture period(between one and 14 days) and the addition ofTGFβ1, BMP-2 or β-glycerophosphate. We trans-planted these dental pulp constructs intoendodontically cleaned and shaped root canals invitro. This information about the adherence ofcells within dental pulp constructs is essential todevelop the new field of regenerative endodontics.
MATERIALS AND METHODS
Tooth cleaning and shaping. We collected 105human teeth from a pre-existing archive ofextracted teeth after receiving approval from theinstitutional review board at Nova SoutheasternUniversity, Fort Lauderdale, Fla. One of us(E.L.G.) examined the teeth under an operatingmicroscope (Global Surgical, St. Louis) to detectpossible fractures, and he excluded those withfractures. Radiographs of each tooth wereassessed to ensure that each one had a single rootcanal. Using a low-speed circular saw (Isomet,Buehler, Lake Bluff, Ill.), one of us (P.E.M.) deco-ronated the teeth near the level of the cemento-enamel junction to provide a root length ofapproximately 16 millimeters.
The dentist (E.L.G.) achieved the root canalworking length by subtracting 1 mm from thelength at which he visualized a 15K-file (DentsplyTulsa Dental, Tulsa, Okla.) at the apicalforamen.23 He cleaned and shaped the teeth usingrotary instruments (ProTaper and ProFile,Dentsply Tulsa Dental). He instrumented the rootcanals using the following sequence of files: SX,S1, S2, F1, F2, F3 and 35/.06. During cleaningand shaping, he irrigated with 1 milliliter of 6 percent sodium hypochlorite (NaClO) solution(Clorox, Oakland, Calif.) after using each instru-
ment size. The clinician used a total of 6 mL ofirrigating solution during the biomechanicalpreparation with the use of 5-mL disposableplastic needles (Ultradent Products, SouthJordan, Utah). This was followed by a one-minuteapplication of 3 mL of 17 percent ethylene-diaminetetraacetic acid (EDTA) (PulpDent,Watertown, Mass.) and by a final flush with 6 mL of 6 percent NaClO.
Disinfection of teeth. After cleaning andshaping the teeth, the dentist (E.L.G.) disinfectedthem by submerging them in 6 percent NaClO forfive minutes. He then washed the specimens insterile saline and rewashed them two times. Heimplanted the dental pulp tissue constructs intothe cleaned and shaped root canals using sterileforceps and endodontic 30 × 21-mm finger plug-gers (Miltex, York, Pa.). The clinician maintainedthe instrumented teeth in Hank’s Balanced SaltSolution (BD Biosciences, San Jose, Calif.) for upto three days at 5oC.
Dental pulp stem cells. The dental pulp stemcells used in this study were SHED teeth,5,6
donated under a material transfer agreementwith the National Institute of Dental and Craniofacial Research (Bethesda, Md.) One of us(P.E.M.) cultured the cells in Dulbecco’s ModifiedEagle’s Medium (DMEM, BD Biosciences). Hemaintained the cell cultures at 37˚C in a humidi-fied atmosphere of 5 percent carbon dioxide, andhe replenished the culture media every secondday for up to 60 days. Confluent cell cultures werecollected by means of trypsinization (0.25 percenttrypsin/2.21 millimolar EDTA, Mediatech, Manassas, Va.).
Implantation of dental pulp tissue con-structs. We investigated two types of 3-D scaf-folds: open-cell polylactic acid (OPLA) and col-lagen scaffolds created from bovine hide (BDBiosciences). The investigator (P.E.M.) sliced eachcylindrical scaffold into two pieces to create ascaffold with an approximate length of 5 mm anda width of 2 mm, as well as an estimated volumeof 0.01195 cubic centimeters. He soaked the scaf-folds in neutral phosphate buffered saline (PBS)and stored them at 5˚C. Twenty-four hours beforecell seeding, he replaced the PBS with DMEM.
Treatment groups 1 through 7. The firsttwo treatment groups were control groups. Thedentist (E.L.G.) cleaned and shaped the rootcanals in group 1 without any scaffolds or cells. Ingroup 2, the second negative control treatmentgroup, we injected SHED × 106 into the cleaned
and shaped root canals of 15 teeth without anyscaffold.5,6 In group 3, one of the experimentaltreatment groups, we incubated the OPLA scaf-fold at 37˚C for 30 minutes before applying thecells to equalize the culture conditions. We cre-ated dental pulp constructs by seeding SHED ×106 in each of the OPLA scaffolds using a sterilemicrosyringe 24 hours before implantation. Usingsterile forceps and endodontic pluggers, the den-tist then implanted the constructs into the rootcanals of 15 cleaned and shaped teeth. Group 4was the same as group 3, except that the scaffoldswere manufactured from bovine collagen. Group 5also was the same as group 3, except that theinvestigator (P.E.M.) injected 50 nanograms ofBMP-2 into the center of each scaffold in 50microliters of 0.1 percent bovine serum albumin(BSA), which was in PBS (pH 7.4).24 Group 6 wasthe same as group 3, except that the investigatorinjected 50 ng of TGFβ1 (Sigma-Aldrich, St.Louis) into the center of each scaffold in 50 µL of0.1 percent BSA, which was in PBS (pH 7.4).Group 7 was the same as group 3, except that heinjected 50 ng of β-glycerophosphate into thecenter of each scaffold in 50 µL of 0.1 percentBSA, which was in PBS (pH 7.4).
The investigator submerged all of the teethcontaining cells, scaffolds and dental pulp con-structs in 1 mL of DMEM culture medium andmaintained them in 24-well culture plates (BDBiosciences) for one, seven or 14 days, accordingto the experimental treatments shown in thetable.
Preparation for scanning electronmicroscopy (SEM). The investigator fixed theteeth by submerging them in a 10 percent neutral-buffered formalin solution at 18oC for 24hours. He then postfixed the teeth in osmiumtetroxide (1 percent volume per volume) for twohours before dehydrating them in a graded seriesof ethanol solutions: 80 percent, 90 percent, 95percent for 15 minutes each, followed by threeconsecutive 10-minute washes in 100 percentethanol.
We removed the teeth from the solutions andplaced them in hexamethyldisilazane for five min-utes to fix the dehydrated specimens. To preparethe teeth for SEM visualization, the investigator(P.E.M.) fractured them into two halves along thelongitudinal axis using a chisel. The teeth weredried on filter paper for 30 minutes. The dentist(E.L.G.) then mounted the tooth specimens ontoaluminum stereoscan stubs with conductive adhe-
sive tabs (Electron Microscopy Services, Hatfield,Pa.). The investigator then sputter-coated thedried-mounted specimens with a 20-30–nanometer thin layer of gold/palladium (model108auto, Cressington Scientific Instruments,Watford, England).
SEM. We viewed the specimens in an SEM(Quanta 200 3D, FEI, Hillsboro, Ore.). Using dig-ital image analysis software, we obtained SEMmicrographs at ×2,000 magnification. We scannedeach of the root canals in its entirety to obtain anoverview of the general surface topography. Inaddition, we visualized cell attachment within thedental pulp constructs and to root canal dentinusing the micrographs. We assessed the effective-ness of the tissue-engineered dental pulp con-structs in adhering to the root canals by using thefollowing semiquantitative criteria:d0: no scaffold;d1: scaffold is not in contact with root canalwalls;d2: scaffold is less than 50 percent in contactwith root canal walls;d3: scaffold is more than 50 percent in contactwith root canal walls.
Data analysis. We analyzed the attachment ofcells within the dental pulp tissue constructs andadherence of the dental pulp constructs to thecleaned and shaped root canals using χ2 statisticsat a significance of 95 percent (Statview, SPSS,Cary, N.C.).
RESULTS
We observed cell attachment for up to 14 daysafter the transplantation of dental pulp tissue
constructs into the cleaned and shaped rootcanals of extracted teeth. Figure 1 shows theempty root canal space after cleaning andshaping, it’s revitalization with a dental pulptissue construct and cell attachment.
The SEM micrographs showed that no cellswere attached to dentin in the negative controlgroup after 14 days of cell culture (Figure 2A,page 462), suggesting that the original pulp tis-sues were removed completely from all teeth.SHED were observed to attach to cleaned andshaped root dentin (Figure 2B), suggesting thatpulp stem cells do not need scaffolds for trans-plantation into teeth. We observed that SHEDattached to the OPLA scaffolds (Figure 2C), thecollagen scaffolds (Figure 2D), the OPLA scaffoldssupplemented with BMP-2 (Figure 2E), the OPLAscaffolds supplemented with TGFβ1 (Figure 2F)and the OPLA scaffolds supplemented with β-glycerophosphate (Figure 2G).
All of the SEM micrographs showed SHEDwith a similar rounded morphology. In addition,dispersal of the cells within the dental pulp tissueconstructs did not appear even, the cells mainlywere grouped together, and we sometimesobserved bare scaffolds. No obvious differenceswere observed with regard to cell activity betweenany of the treatment groups, with the exception ofthe negative control group. The SEM micrographssuggest that adherence of the dental pulp con-structs was best in the coronal aspect of teeth andless optimal in the middle and apical thirds of theroot canal.
The adherence of dental pulp constructs tocleaned and shaped root canals was similar for
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Testing schedule for pulp tissue–engineered constructs.TREATMENTGROUP
CELL TYPE 3-D SCAFFOLD TYPE GROWTH FACTORS
NUMBER OF SAMPLES
Day 1 Day 7 Day 14
1 None None (control) None 5 5 5
2 SHED* None (control) None 5 5 5
3 SHED Open-cell polylactic acid None 5 5 5
4 SHED Collagen None 5 5 5
5 SHED Open-cell polylactic acid Bone morphogenic protein-2
the various treatment groups (χ2, P < .06). TheOPLA constructs are degradable, and after 14days of cell culture, the constructs treated withBMP-2 and TGFβ1 appeared to be slightly less incontact with the root canal walls than were theconstructs in the OPLA group (Figure 3, page463), although the length of the cell culture perioddid not appear to influence construct attachmentto dentin (χ2, P < .06). All of the dental pulp con-structs had some contact with the root canals,although the contact was frequently less than 50percent. The OPLA scaffolds appeared to attachmore completely with the root canal dentin thandid the collagen scaffolds (Figure 3).
DISCUSSION
Regenerative endodontic procedures are biologi-cally based procedures designed to replace dam-aged structures including dentin and root struc-tures, as well as cells of the pulp-dentin complex.The objectives of regenerative endodontic pro-cedures are to regenerate pulplike tissue, ideally,the pulp-dentin complex among other dentalstructures.22 Some authors25,26 have suggestedthat the revascularization of the adult (closed)apex may be accomplished by opening up thetooth apex to approximately 1 mm in diameter toallow systemic bleeding into root canals. The ideafor this therapy came from the pioneering studiesof Nygaard-Ostby and Hørsted27,28 in the 1970s.They reported new connective-tissue formation inthe root canal after total pulpectomy and partialroot filling.27,28
Renewed interest in the ability of new tissues
to form within root canals was stimulated by acase report by Banchs and Trope.25 They reportedradiographic evidence of tooth healing in an 11-year-old boy where the root canal had been disin-fected. The authors acknowledged that they didnot know if the vital tissue seen in the radiographwas pulp tissue or if this treatment would beeffective in the long term. Nevertheless, theseobservations provide some evidence that pulprevascularization can be accomplished clinically.To make progress in the field of regenerativeendodontics and to supplement the clinical pulprevascularization studies, we have examined thetransplantation of dental pulp tissue constructs,using various treatments, into in vitro cleanedand shaped root canals. Further research in thisfield using the latest tissue-engineering tech-nology is necessary to optimize the creation ofdental pulp tissue constructs by using stem cells,growth factors and 3-D tissue-engineering scaf-folds to be incorporated into future endodontictreatment.
Cell attachment and activity. All of thedental pulp tissue constructs created in this studyand inserted into cleaned and shaped root canalsmaintained cell attachment, suggesting that theculture conditions supported the continuedgrowth of the cells in vitro. However, the additionof the biomolecules and growth factors TGFβ1,BMP-2 and β-glycerophosphate did not appear tobenefit cell attachment or cell activity, suggestingthat they may be of limited usefulness for in vitrotissue construct cell culturing. Biomoleculesmight have more beneficial effects in monolayer
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Figure 1. Scanning electron micrographs of a tissue-engineered pulp construct inside a shaped and cleaned root canal.
The reason for the lack of cell responsiveness tobiomolecule activity requires further investiga-tion, although it seems likely that the porousstructure of the scaffolds allowed the biomole-cules to disperse into the culture medium, ratherthan be retained on the scaffolds where cells wereattached, thereby diluting the biomolecules awayfrom the cells. To avoid this problem in futurestudies, investigators should bind growth factorsand biomolecules to the scaffold to optimize theireffect on stem cells. To identify biomolecules withthe potential to promote cell attachment, investi-gators need to examine the extracellular matrix
interactions between stem cells and tooth dentin.SEM micrographs. The SEM micrographs in
this study showed that no cells were attached todentin in the negative control groups. This sug-gests that the original pulp tissues were removedcompletely from all teeth and, therefore, that allof the cells visualized ultrastructurally wereSHED that were added in the laboratory. Ourobservation that SHED attached to cleaned andshaped root dentin might suggest that pulp stemcells do not need scaffolds for transplantation intoteeth, because it appears that pulp stem cellsdelivered to root canals attach naturally tocleaned root dentin.
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Figure 2. Scanning electron micrographs of the coronal regions of dentin, cells and tissue-engineered pulp constructs.
However, we must consider the complete revi-talization of the root canal. In the absence of ascaffold to support cell attachment, it may beimpossible for stem cells to repopulate the core ofroot canals, suggesting that scaffolds and tissueconstructs or blood clots are necessary to accom-plish endodontic regeneration. In our study, weused a root canal working length that was 1 mmshort of the apical foramen to be consistent with acommon published methodology,23 as well as toprevent the escape of nutrients and SHED fromthe tooth during in vitro culture.
Potential clinical success. The clinical suc-cess of dental pulp constructs likely will dependon their integration with the body and revascular-ization. Therefore, it will be essential to open thetooth apex to allow systemic bleeding into rootcanals.25-28 During the in vitro instrumentation ofteeth, we used small quantities of irrigants;larger quantities likely will be needed for in vivostudies. In addition, we submerged the teeth inNaClO to ensure that they were completely disin-fected before placing the dental pulp implants.Disinfection of root canals to minimize the pres-ence of bacteria may be essential to maintain thesurvival and integration of dental pulp con-structs. In clinical studies, researchers may use a
tribiotic paste30 to ensure that the root canal isdisinfected adequately before using the blood clotmethod of revascularization of root canals,25 per-forming regenerative endodontic therapy22 orimplanting dental pulp constructs.
Cell dispersal. Ideally, the pulp stem cellswould seed evenly throughout the dental pulptissue constructs, as is observed in the dentalpulp tissue. The asymmetric dispersal of cellswithin the dental pulp constructs created in thisstudy is not a new problem; it has been observedin many tissue-engineering studies.31 It is likelythat once the dental pulp constructs are trans-planted into animals or used in clinical studies,the nutrients and vascularization available mayhelp make cell attachment more uniform.32 There-fore, the dispersal of cells within the dental pulpconstructs appears to be a function of the in vitroconditions, which can be resolved once they areused clinically.
The advantage of implanting dental pulp con-structs into cleaned and shaped root canals overthe blood clot revascularization technique25-28 isthat the source of the cells regenerating thereplacement pulp tissue is endodontic in origin.In contrast, after blood clot revascularization, the
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2.5
1.5
.5
None SHED OPLASHED
BovineCollagen
SHED
OPLABMP-2SHED
OPLATGFβ1SHED
OPLAβ-GlycerophosphateSHED
3 (Scaffold Is More Than50% in Contact With
Root Canal Walls)
2 (Scaffold Is Less Than50% in Contact With
Root Canal Walls)
1 (No Scaffold Contact With Root Canal Walls)
0 (No Scaffold)
TREATMENT GROUP
AT
TA
CH
MEN
T O
F D
EN
TA
L P
ULP
TIS
SU
ECO
NST
RU
CT
S T
O R
OO
T C
AN
ALS Time of Cell Culture
One Day
Seven Days
14 Days
Figure 3. Attachment of a dental pulp construct to root dentin. SHED: Stem cells from human exfoliated deciduous teeth. OPLA: Open-cellpolylactic acid. BMP: Bone morphogenic protein. TGFβ1: Transforming growth factor β1. The bars represent the standard deviation of themean.
origins of the cells regenerating new tissues inthe root canals have not been identified.25-28 Thelikely origins of the cells may be remnant pulpstem cells in lateral canals, stem cells from theperiodontal ligament or stem cells from the rootapical papilla.33
The adherence of dental pulp constructs tocleaned and shaped root canals was similar for thevarious treatment groups, and the length of timeof the cell culture did not appear to influence con-struct attachment to dentin. After 14 days, dentalpulp constructs treated with BMP-2 and TGFβ1appeared to be in slightly less contact with theroot canal walls than were the other dental pulpconstructs, which may have been caused byincreasing cell activity to digest the degradableOPLA scaffold. Longer-term studies are requiredto investigate the relationships between bioactivemolecules, stem cell activity and scaffold degradation.
Attachment to root canal surfaces. Theattachment of dental pulp constructs to root canalsurfaces in this study was less impressive thanwe had hoped for. The goal of creating functionaldental pulp constructs through pulp stem cellattachment to tissue-engineering scaffolds, aswell as achieving their complete attachment toroot canal surfaces, still appears to be a substan-tial problem. We must accept that to have dentalpulp constructs growing in root canals withoutfunctional connection to the dentin body is rathermeaningless, as a well-placed root filling wouldaccomplish the same. The ability to regenerate areplacement vital pulp attached to the circulatorysystem and the old dentin, as well as produce anew dentin matrix, does not appear to be an easytask to accomplish.
CONCLUSIONS
Our study provides evidence of the proof-of-concept for the transplantation of dental pulp con-structs into cleaned and shaped root canals. How-ever, the occurrence of SHED on parts of the rootcanal wall is not a conclusive presumption for thepotential of this technique to revascularize andrevitalize cleaned and shaped root canals. Follow-up tissue-engineering studies are needed to helpmake regenerative endodontics a reality.
Improvements to the design of dental pulptissue constructs will make their adherence toroot canal dentin more complete. We noted thatthe dental pulp tissue constructs adhered morecompletely to the coronal aspects of the root canal
and less completely to the middle and apicalaspects. This likely was caused by the increasingcomplexity of root canal anatomy toward the apexand the physical constraints of the scaffoldmaterials, as well as the placement method. Toimprove the ability of dental pulp constructs toadhere to root canal walls, it seems that the idealscaffold design is in the same shape as gutta-percha cones.
We used single-canal teeth and cylindrical scaf-folds in an attempt to simplify the transplanta-tion process. A more complex root canal anatomywill require more complex scaffolds or the use ofmore flexible scaffolds to perform regenerativeendodontics. The potential benefit of trans-planting tissue-engineered dental pulp into rootcanals is that it will revitalize teeth, therebyrestoring the natural state of the tooth so that itcan be sensitive, repair itself and respond todental injuries. ■
Disclosure: None of the authors reported any disclosures.
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