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J Am Dent Assoc, Vol 139, No 4, 457-465. © 2008 American Dental Association

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	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
Background. The authors conducted an ultrastructural scanning electron microscopic (SEM) investigation of tissue-engineered pulp constructs implanted within endodontically treated teeth.

Methods. Stem cells from human exfoliated deciduous teeth were seeded on a synthetic open-cell D,D-L,L-polylactic acid scaffold with or without the addition of bone morphogenic protein-2 and transforming growth factor β1 to create pulp tissue constructs. The pulp constructs were implanted into 105 extracted human premolar teeth with a single root canal that had been cleaned and shaped by using rotary instrumentation in a crown-down manner to ISO size no. 35.

Results. An ultrastructural examination of the SEM micrographs at x2,000 magnification revealed cell adherence within all of the pulp constructs, with little difference between the scaffold types or with the addition of growth factors.

Conclusions. These results support the proof-of-concept that it is possible to implant tissue-engineered pulp constructs into teeth after cleaning and shaping.

Clinical Implications. Future regenerative endodontic treatment may involve the cleaning and shaping of root canals followed by the implantation of vital dental pulp tissue constructs created in the laboratory.

Key Words: Stem cells; root canal; endodontics; pulpitis; dental pulp tissue constructs

Abbreviations: BMP: Bone morphogenic protein. • BSA: Bovine serum albumin. • DMEM: Dulbecco’s Modified Eagle’s Medium (BD Biosciences, San Jose, Calif.). • EDTA: Ethylenediaminetetraacetic acid. • NaClO: Sodium hypochlorite. • OPLA: Open-cell polylactic acid. • PBS: Phosphate buffered saline. • SEM: Scanning electron microscopy. • SHED: Stem cells from human exfoliated deciduous teeth. • TGFβ: Transforming growth factor β.

The most valuable cells for regenerative dentistry are stem cells.¹ A stem cell is commonly defined as a cell that has the ability to divide continuously and produce progeny cells that differentiate (develop) into various other types of cells or tissues.² The dental pulp contains a stem cell population known as pulp stem cells^3,4 or, in the case of immature teeth, stem cells from human exfoliated deciduous teeth (SHED).^5,6

To create a practical endodontic tissue-engineering therapy, investigators must organize pulp stem cells into a three-dimensional structure using tissue-engineering scaffolds. Several studies of dental pulp cells seeded on 3-D scaffolds have been completed. Recently, Zhang and colleagues⁷ observed successful dental pulp stem cell growth on spongeous collagen, porous ceramic and fibrous titanium mesh scaffolds that were implanted into nude mice. Hee and colleagues⁸ found that bio-ceramic calcium phosphate tissue scaffolds appear more optimal for bone regeneration in comparison with polymer scaffolds. However, biodegradable polymer scaffolds manufactured from the same biomaterials used in surgical dissolvable sutures^9,10 have an advantage in that these biomaterials have been proven to be biocompatible according to screening assays approved by the U.S. Food and Drug Administration.¹¹

Several studies have suggested that polymer scaffolds hold the most promise for creating replacement tissues through tissue engineering. Human dental pulp and gingival fibroblasts adhere, proliferate and produce extracellular matrix when seeded on polymer scaffolds in vitro.¹² Researchers also have used polymer scaffolds to bioengineer tooth tissues from porcine¹³ and rodent tooth bud cells,¹⁴ but they have not yet seeded polymer scaffolds with human dental pulp stem cells or SHED to be investigated for dental pulp tissue engineering.

The seeding of cells on tissue engineering scaffolds is known as "creating a tissue construct."¹⁵ To promote the formation of higher-ordered tissue structures, tissue constructs are maintained in cell culture in the presence of growth factors or bioactive molecules. Growth factors, especially those of the transforming growth factor β (TGFβ) family, are important in cellular signaling for odontoblast differentiation and stimulation of dentin matrix secretion. These growth factors are secreted by odontoblasts and are deposited within the dentin matrix, where they remain protected in an active form through interaction with other components of the dentin matrix.¹⁶ The addition of purified dentin protein fractions has stimulated an increase in tertiary dentin matrix secretion, suggesting that TGFβ1 is involved in injury-signaling and tooth-healing reactions.¹⁷

Another important growth factor in tooth development and regeneration is bone morphogenic proteins (BMPs).¹⁸ Recombinant human BMP-2 stimulates differentiation of adult pulp stem cells into an odontoblastoid morphology in culture.¹⁹ Researchers have demonstrated the similar effects of TGFβ1-3 and BMP-7 in cultured tooth slices.²⁰ Recombinant BMP-2, -4 and -7 induce reparative dentin in vivo.²¹ These studies^18–21 suggest that the addition of growth factors to the culture media may promote the formation of dental pulp tissue constructs. However, it is unclear if BMP-2 or TFGβ1 is optimal, and it is not known what effects these growth factors have on tissue-engineered constructs.

Numerous studies have demonstrated that cells can attach to and grow on dentin surfaces, while other studies have shown that cells can attach to tissue-engineering scaffolds.²² However, no previous studies, to our knowledge, have investigated the potential of SHED to create dental pulp constructs within human cleaned and shaped root canals, which has been suggested as a possible future endodontic regenerative treatment.²²

Therefore, the purpose of this study was to examine how SHED attachment within a porous polymer scaffold can be used to create a dental pulp tissue construct. The variables to be tested included the longevity of the cell culture period (between one and 14 days) and the addition of TGFβ1, BMP-2 or β-glycerophosphate. We transplanted these dental pulp constructs into endodontically cleaned and shaped root canals in vitro. This information about the adherence of cells within dental pulp constructs is essential to develop the new field of regenerative endodontics.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
Tooth cleaning and shaping. We collected 105 human teeth from a pre-existing archive of extracted teeth after receiving approval from the institutional review board at Nova Southeastern University, Fort Lauderdale, Fla. One of us (E.L.G.) examined the teeth under an operating microscope (Global Surgical, St. Louis) to detect possible fractures, and he excluded those with fractures. Radiographs of each tooth were assessed to ensure that each one had a single root canal. Using a low-speed circular saw (Isomet, Buehler, Lake Bluff, Ill.), one of us (P.E.M.) decoronated the teeth near the level of the cemento-enamel junction to provide a root length of approximately 16 millimeters.

The dentist (E.L.G.) achieved the root canal working length by subtracting 1 mm from the length at which he visualized a 15K-file (Dentsply Tulsa Dental, Tulsa, Okla.) at the apical foramen.²³ He cleaned and shaped the teeth using rotary instruments (ProTaper and ProFile, Dentsply Tulsa Dental). He instrumented the root canals using the following sequence of files: SX, S1, S2, F1, F2, F3 and 35/.06. During cleaning and shaping, he irrigated with 1 milliliter of 6 percent sodium hypochlorite (NaClO) solution (Clorox, Oakland, Calif.) after using each instrument size. The clinician used a total of 6 mL of irrigating solution during the biomechanical preparation with the use of 5-mL disposable plastic needles (Ultradent Products, South Jordan, Utah). This was followed by a one-minute application 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 and shaping the teeth, the dentist (E.L.G.) disinfected them by submerging them in 6 percent NaClO for five minutes. He then washed the specimens in sterile saline and rewashed them two times. He implanted the dental pulp tissue constructs into the cleaned and shaped root canals using sterile forceps and endodontic 30 x 21-mm finger pluggers (Miltex, York, Pa.). The clinician maintained the instrumented teeth in Hank’s Balanced Salt Solution (BD Biosciences, San Jose, Calif.) for up to three days at 5^oC.

Dental pulp stem cells. The dental pulp stem cells used in this study were SHED teeth,^5,6 donated under a material transfer agreement with the National Institute of Dental and Craniofacial Research (Bethesda, Md.) One of us (P.E.M.) cultured the cells in Dulbecco’s Modified Eagle’s Medium (DMEM, BD Biosciences). He maintained the cell cultures at 37°C in a humidified atmosphere of 5 percent carbon dioxide, and he replenished the culture media every second day for up to 60 days. Confluent cell cultures were collected by means of trypsinization (0.25 percent trypsin/2.21 millimolar EDTA, Mediatech, Manassas, Va.).

Implantation of dental pulp tissue constructs. We investigated two types of 3-D scaffolds: open-cell polylactic acid (OPLA) and collagen scaffolds created from bovine hide (BD Biosciences). The investigator (P.E.M.) sliced each cylindrical scaffold into two pieces to create a scaffold with an approximate length of 5 mm and a width of 2 mm, as well as an estimated volume of 0.01195 cubic centimeters. He soaked the scaffolds in neutral phosphate buffered saline (PBS) and stored them at 5°C. Twenty-four hours before cell seeding, he replaced the PBS with DMEM.

Treatment groups 1 through 7. The first two treatment groups were control groups. The dentist (E.L.G.) cleaned and shaped the root canals in group 1 without any scaffolds or cells. In group 2, the second negative control treatment group, we injected SHED x 10⁶ into the cleaned and shaped root canals of 15 teeth without any scaffold.^5,6 In group 3, one of the experimental treatment groups, we incubated the OPLA scaffold at 37°C for 30 minutes before applying the cells to equalize the culture conditions. We created dental pulp constructs by seeding SHED x 10⁶ in each of the OPLA scaffolds using a sterile microsyringe 24 hours before implantation. Using sterile forceps and endodontic pluggers, the dentist then implanted the constructs into the root canals of 15 cleaned and shaped teeth. Group 4 was the same as group 3, except that the scaffolds were manufactured from bovine collagen. Group 5 also was the same as group 3, except that the investigator (P.E.M.) injected 50 nanograms of BMP-2 into the center of each scaffold in 50 microliters of 0.1 percent bovine serum albumin (BSA), which was in PBS (pH 7.4).²⁴ Group 6 was the same as group 3, except that the investigator injected 50 ng of TGFβ1 (Sigma-Aldrich, St. Louis) into the center of each scaffold in 50 µL of 0.1 percent BSA, which was in PBS (pH 7.4). Group 7 was the same as group 3, except that he injected 50 ng of β-glycerophosphate into the center of each scaffold in 50 µL of 0.1 percent BSA, which was in PBS (pH 7.4).

The investigator submerged all of the teeth containing cells, scaffolds and dental pulp constructs in 1 mL of DMEM culture medium and maintained them in 24-well culture plates (BD Biosciences) for one, seven or 14 days, according to the experimental treatments shown in the table.

 
Preparation for scanning electron microscopy (SEM). The investigator fixed the teeth by submerging them in a 10 percent neutral-buffered formalin solution at 18^oC for 24 hours. He then postfixed the teeth in osmium tetroxide (1 percent volume per volume) for two hours before dehydrating them in a graded series of ethanol solutions: 80 percent, 90 percent, 95 percent for 15 minutes each, followed by three consecutive 10-minute washes in 100 percent ethanol.

We removed the teeth from the solutions and placed them in hexamethyldisilazane for five minutes to fix the dehydrated specimens. To prepare the teeth for SEM visualization, the investigator (P.E.M.) fractured them into two halves along the longitudinal axis using a chisel. The teeth were dried on filter paper for 30 minutes. The dentist (E.L.G.) then mounted the tooth specimens onto aluminum stereoscan stubs with conductive adhesive tabs (Electron Microscopy Services, Hatfield, Pa.). The investigator then sputter-coated the dried-mounted specimens with a 20–30–nanometer thin layer of gold/palladium (model 108auto, Cressington Scientific Instruments, Watford, England).

SEM. We viewed the specimens in an SEM (Quanta 200 3D, FEI, Hillsboro, Ore.). Using digital image analysis software, we obtained SEM micrographs at x2,000 magnification. We scanned each of the root canals in its entirety to obtain an overview of the general surface topography. In addition, we visualized cell attachment within the dental pulp constructs and to root canal dentin using the micrographs. We assessed the effectiveness of the tissue-engineered dental pulp constructs in adhering to the root canals by using the following semiquantitative criteria:

– 0: no scaffold;

– 1: scaffold is not in contact with root canal walls;

– 2: scaffold is less than 50 percent in contact with root canal walls;

– 3: scaffold is more than 50 percent in contact with root canal walls.

Data analysis. We analyzed the attachment of cells within the dental pulp tissue constructs and adherence of the dental pulp constructs to the cleaned and shaped root canals using ² statistics at a significance of 95 percent (Statview, SPSS, Cary, N.C.).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
We observed cell attachment for up to 14 days after the transplantation of dental pulp tissue constructs into the cleaned and shaped root canals of extracted teeth. Figure 1 shows the empty root canal space after cleaning and shaping, it’s revitalization with a dental pulp tissue construct and cell attachment.

View larger version (54K): [in this window] [in a new window]   Figure 1. Scanning electron micrographs of a tissue-engineered pulp construct inside a shaped and cleaned root canal.

 
The SEM micrographs showed that no cells were attached to dentin in the negative control group after 14 days of cell culture (Figure 2A, page 462), suggesting that the original pulp tissues were removed completely from all teeth. SHED were observed to attach to cleaned and shaped root dentin (Figure 2B), suggesting that pulp stem cells do not need scaffolds for transplantation into teeth. We observed that SHED attached to the OPLA scaffolds (Figure 2C), the collagen scaffolds (Figure 2D), the OPLA scaffolds supplemented with BMP-2 (Figure 2E), the OPLA scaffolds supplemented with TGFβ1 (Figure 2F) and the OPLA scaffolds supplemented with β-glycerophosphate (Figure 2G).

View larger version (90K): [in this window] [in a new window]   Figure 2. Scanning electron micrographs of the coronal regions of dentin, cells and tissue-engineered pulp constructs.

 
All of the SEM micrographs showed SHED with a similar rounded morphology. In addition, dispersal of the cells within the dental pulp tissue constructs did not appear even, the cells mainly were grouped together, and we sometimes observed bare scaffolds. No obvious differences were observed with regard to cell activity between any of the treatment groups, with the exception of the negative control group. The SEM micrographs suggest that adherence of the dental pulp constructs was best in the coronal aspect of teeth and less optimal in the middle and apical thirds of the root canal.

The adherence of dental pulp constructs to cleaned and shaped root canals was similar for the various treatment groups (², P < .06). The OPLA constructs are degradable, and after 14 days of cell culture, the constructs treated with BMP-2 and TGFβ1 appeared to be slightly less in contact with the root canal walls than were the constructs in the OPLA group (Figure 3, page 463), although the length of the cell culture period did not appear to influence construct attachment to dentin (², P < .06). All of the dental pulp constructs had some contact with the root canals, although the contact was frequently less than 50 percent. The OPLA scaffolds appeared to attach more completely with the root canal dentin than did the collagen scaffolds (Figure 3).

View larger version (55K): [in this window] [in a new window]   Figure 3. Attachment of a dental pulp construct to root dentin. SHED: Stem cells from human exfoliated deciduous teeth. OPLA: Open-cell polylactic acid. BMP: Bone morphogenic protein. TGFβ1: Transforming growth factor β1. The bars represent the standard deviation of the mean.

 

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
Regenerative endodontic procedures are biologically based procedures designed to replace damaged structures including dentin and root structures, as well as cells of the pulp-dentin complex. The objectives of regenerative endodontic procedures are to regenerate pulplike tissue, ideally, the pulp-dentin complex among other dental structures.²² Some authors^25,26 have suggested that the revascularization of the adult (closed) apex may be accomplished by opening up the tooth apex to approximately 1 mm in diameter to allow systemic bleeding into root canals. The idea for this therapy came from the pioneering studies of Nygaard-Ostby and Hørsted^27,28 in the 1970s. They reported new connective-tissue formation in the root canal after total pulpectomy and partial root filling.^27,28

Renewed interest in the ability of new tissues to form within root canals was stimulated by a case report by Banchs and Trope.²⁵ They reported radiographic evidence of tooth healing in an 11-year-old boy where the root canal had been disinfected. The authors acknowledged that they did not know if the vital tissue seen in the radiograph was pulp tissue or if this treatment would be effective in the long term. Nevertheless, these observations provide some evidence that pulp revascularization can be accomplished clinically. To make progress in the field of regenerative endodontics and to supplement the clinical pulp revascularization studies, we have examined the transplantation of dental pulp tissue constructs, using various treatments, into in vitro cleaned and shaped root canals. Further research in this field using the latest tissue-engineering technology is necessary to optimize the creation of dental pulp tissue constructs by using stem cells, growth factors and 3-D tissue-engineering scaffolds to be incorporated into future endodontic treatment.

Cell attachment and activity. All of the dental pulp tissue constructs created in this study and inserted into cleaned and shaped root canals maintained cell attachment, suggesting that the culture conditions supported the continued growth of the cells in vitro. However, the addition of the biomolecules and growth factors TGFβ1, BMP-2 and β-glycerophosphate did not appear to benefit cell attachment or cell activity, suggesting that they may be of limited usefulness for in vitro tissue construct cell culturing. Biomolecules might have more beneficial effects in monolayer cell cultures²⁹ and in vivo animal experiments.¹⁸ The reason for the lack of cell responsiveness to biomolecule activity requires further investigation, although it seems likely that the porous structure of the scaffolds allowed the biomolecules to disperse into the culture medium, rather than be retained on the scaffolds where cells were attached, thereby diluting the biomolecules away from the cells. To avoid this problem in future studies, investigators should bind growth factors and biomolecules to the scaffold to optimize their effect on stem cells. To identify biomolecules with the potential to promote cell attachment, investigators 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 to dentin in the negative control groups. This suggests that the original pulp tissues were removed completely from all teeth and, therefore, that all of the cells visualized ultrastructurally were SHED that were added in the laboratory. Our observation that SHED attached to cleaned and shaped root dentin might suggest that pulp stem cells do not need scaffolds for transplantation into teeth, because it appears that pulp stem cells delivered to root canals attach naturally to cleaned root dentin.

However, we must consider the complete revitalization of the root canal. In the absence of a scaffold to support cell attachment, it may be impossible for stem cells to repopulate the core of root canals, suggesting that scaffolds and tissue constructs or blood clots are necessary to accomplish endodontic regeneration. In our study, we used a root canal working length that was 1 mm short of the apical foramen to be consistent with a common published methodology,²³ as well as to prevent the escape of nutrients and SHED from the tooth during in vitro culture.

Potential clinical success. The clinical success of dental pulp constructs likely will depend on their integration with the body and revascularization. Therefore, it will be essential to open the tooth apex to allow systemic bleeding into root canals.^25–28 During the in vitro instrumentation of teeth, we used small quantities of irrigants; larger quantities likely will be needed for in vivo studies. In addition, we submerged the teeth in NaClO to ensure that they were completely disinfected before placing the dental pulp implants. Disinfection of root canals to minimize the presence of bacteria may be essential to maintain the survival and integration of dental pulp constructs. In clinical studies, researchers may use a tribiotic paste³⁰ to ensure that the root canal is disinfected adequately before using the blood clot method of revascularization of root canals,²⁵ performing regenerative endodontic therapy²² or implanting dental pulp constructs.

Cell dispersal. Ideally, the pulp stem cells would seed evenly throughout the dental pulp tissue constructs, as is observed in the dental pulp tissue. The asymmetric dispersal of cells within the dental pulp constructs created in this study is not a new problem; it has been observed in many tissue-engineering studies.³¹ It is likely that once the dental pulp constructs are transplanted into animals or used in clinical studies, the nutrients and vascularization available may help make cell attachment more uniform.³² Therefore, the dispersal of cells within the dental pulp constructs appears to be a function of the in vitro conditions, which can be resolved once they are used clinically.

The advantage of implanting dental pulp constructs into cleaned and shaped root canals over the blood clot revascularization technique^25–28 is that the source of the cells regenerating the replacement pulp tissue is endodontic in origin. In contrast, after blood clot revascularization, the origins of the cells regenerating new tissues in the root canals have not been identified.^25–28 The likely origins of the cells may be remnant pulp stem cells in lateral canals, stem cells from the periodontal ligament or stem cells from the root apical papilla.³³

The adherence of dental pulp constructs to cleaned and shaped root canals was similar for the various treatment groups, and the length of time of the cell culture did not appear to influence construct attachment to dentin. After 14 days, dental pulp constructs treated with BMP-2 and TGFβ1 appeared to be in slightly less contact with the root canal walls than were the other dental pulp constructs, which may have been caused by increasing cell activity to digest the degradable OPLA scaffold. Longer-term studies are required to investigate the relationships between bioactive molecules, stem cell activity and scaffold degradation.

Attachment to root canal surfaces. The attachment of dental pulp constructs to root canal surfaces in this study was less impressive than we had hoped for. The goal of creating functional dental pulp constructs through pulp stem cell attachment to tissue-engineering scaffolds, as well as achieving their complete attachment to root canal surfaces, still appears to be a substantial problem. We must accept that to have dental pulp constructs growing in root canals without functional connection to the dentin body is rather meaningless, as a well-placed root filling would accomplish the same. The ability to regenerate a replacement vital pulp attached to the circulatory system and the old dentin, as well as produce a new dentin matrix, does not appear to be an easy task to accomplish.

	CONCLUSIONS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
Our study provides evidence of the proof-of-concept for the transplantation of dental pulp constructs into cleaned and shaped root canals. However, the occurrence of SHED on parts of the root canal wall is not a conclusive presumption for the potential of this technique to revascularize and revitalize cleaned and shaped root canals. Follow-up tissue-engineering studies are needed to help make regenerative endodontics a reality.

Improvements to the design of dental pulp tissue constructs will make their adherence to root canal dentin more complete. We noted that the dental pulp tissue constructs adhered more completely to the coronal aspects of the root canal and less completely to the middle and apical aspects. This likely was caused by the increasing complexity of root canal anatomy toward the apex and the physical constraints of the scaffold materials, as well as the placement method. To improve the ability of dental pulp constructs to adhere to root canal walls, it seems that the ideal scaffold design is in the same shape as gutta-percha cones.

We used single-canal teeth and cylindrical scaffolds in an attempt to simplify the transplantation process. A more complex root canal anatomy will require more complex scaffolds or the use of more flexible scaffolds to perform regenerative endodontics. The potential benefit of transplanting tissue-engineered dental pulp into root canals is that it will revitalize teeth, thereby restoring the natural state of the tooth so that it can be sensitive, repair itself and respond to dental injuries.

	FOOTNOTES

Dr. Murray is an associate professor, Department of Endodontics, College of Dental Medicine, Nova Southeastern University, 3200 S. University Drive, Fort Lauderdale, Fla. 33328-2018, e-mail "petemurr{at}nova.edu". Address reprint requests to Dr. Murray.

Dr. Namerow is an associate professor and chair, Department of Endodontics, College of Dental Medicine, Nova Southeastern University, Fort Lauderdale, Fla.

Dr. Kuttler is an associate professor and assistant dean for postgraduate education, College of Dental Medicine, Nova Southeastern University, Fort Lauderdale, Fla.

Dr. Garcia-Godoy is a professor and associate dean for research, College of Dental Medicine, Nova Southeastern University, Fort Lauderdale, Fla.

Disclosure: None of the authors reported any disclosures.

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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 

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