Basic ResearchBiology

Cell Survival within Pulp and Periodontal Constructs

Matthew Gebhardt, DDS,* Peter E. Murray, PhD, Kenneth N. Namerow, DDS, Sergio Kuttler, DDS, and Franklin Garcia-Godoy, DDS, MS
Abstract
The purpose of this study was to measure cell survival and degradation within tissue-engineered dental constructs. Dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PLSCs) were seeded on three types of tissue engineering scaffolds: a synthetic open-cell D,D-L,L-polylactic acid (polymer) scaffold, a bovine collagen scaffold (collagen), and a calcium phosphate bioceramic (calcium phosphate) scaffold. The dental pulp and periodontal constructs (n 144) were maintained in cell culture for between 3 and 14 days. The cell survival and degradation within the constructs were measured using histologic criteria. The DPSC and PLSC survival was optimal in the polymer and collagen constructs but not the calcium phosphate constructs, especially over longer time periods. These in vitro results suggest that both the polymer and collagen scaffolds and the DPSCs and PLSCs can be combined to create pulp and periodontal constructs for use in future regenerative dental treatments. (J Endod 2009;35:63 66)

Key Words
Endodontics, regenerative, scaffolds, stem cells, tissue engineering

From *Private practice, Medford, OR, and the Department of Endodontics, College of Dental Medicine, Nova Southeastern University, Fort Lauderdale, Florida. Supported by the American Association of Endodontists Foundation and NSU Health Professions Divisions Faculty Development Grants. Address requests for reprints to Dr Peter E. Murray, Department of Endodontics, College of Dental Medicine, Nova Southeastern University, 3200 South University Drive, Fort Lauderdale, FL 33328-2018. E-mail address: petemurr@nova. edu. 0099-2399/$0 - see front matter Copyright 2008 American Association of Endodontists. doi:10.1016/j.joen.2008.09.020

he regeneration and replacement of dental tissues has been a goal of dentists for decades, but this goal has been limited by the technology available to practitioners, in addition to the lack of healing potential of diseased, traumatized, or missing patient dental tissues. Since the late 1960s, iliac cancellous bone grafts were used to regenerate furcations, dehiscences, and intraosseous periodontal defects (13). Several years later, tissue regeneration in the root canal was observed after total pulpectomy and partial root filling (4, 5). Recently, some case reports have shown the potential of necrotic teeth to revascularize after disinfection with triple antibiotic paste and after establishing bleeding in the root canal (6 8). Advances in stem cell purification, cell culture technology, and the design of biomaterial scaffolds may allow the de novo creation of tissues in the laboratory. These tissue-engineering advances have the potential to create pulp and periodontal constructs, which may allow future dentists to replace or regenerate diseased, traumatized, or missing pulp and periodontal tissues. In response to the potential advances in endodontic treatments, the new field of regenerative endodontics has been created (9). The most valuable cells to accomplish regenerative endodontics are dental pulp stem cells (DPSCs) from human exfoliated deciduous teeth (10, 11) and human periodontal ligament stem cells (PLSCs) (12). DPSCs may also be obtained from wisdom teeth. To create a practical regenerative endodontic therapy, these stem cells must be organized into a three-dimensional structure using tissue-engineering scaffold materials (9) to create a tissue construct (13). Collagen scaffolds can be created from hydrochloric acid and acetic acid (14). Collagen scaffolds are popular in the scientific literature because they are structurally similar to natural human tissues, can support cell growth, and can be transplanted into animals with few immunologic reactions to regenerate tissue function (15). The transplantation of constructs, created by combining DPSCs with collagen scaffolds and dentin matrix protein-1, has already proved to be successful at regenerating dental pulp-like tissue in simulated furcal perforation sites in nude mice (16). A previous attempt to create dental pulp tissues by the subcutaneous transplantation of constructs created by seeding DPSCs on collagen, ceramic, and titanium mesh scaffolds into nude mice appeared to cause the creation of connective tissues (17). Synthetic polymer scaffolds are generally fabricated from the polyglycolic or polylactic acid materials similar to surgical sutures (18). A major advantage of these scaffolds is their biocompatibility and approval for surgical use by the food and drug association (19). Polymer scaffolds have already been used to bioengineer tooth tissues from porcine (20) and rodent tooth bud cells (21) as well as periodontal ligament constructs (22). An ultrastructural study showed the implantation of dental pulp constructs into cleaned and shaped root canals as a putative future regenerative endodontic therapy (23). Calcium phosphate cement can be used as a root canalfilling material (24), and calcium phosphate scaffolds have proved to be successful for the cellular proliferation and osteogenic differentiation of human bone marrow stromal cells (25). However, it remains uncertain which type of scaffold material, collagen, polymer, or calcium phosphate, will provide the optimal survival substrate for DPSCs and PLSCs. The Food and Drug Administration has recently approved collagen and polymer scaffolds for peripheral and cranial nerve repair (26), which suggests that these scaffolds could be approved by the FDA for dental repair in the future. The purpose of this study was to measure the survival of DPSCs and PLSCs when maintained in cell culture on collagen, polymer, and calcium phosphate scaffolds to create de novo dental pulp and periodontal constructs.

JOE Volume 35, Number 1, January 2009

Cell Survival within Periodontal Constructs

63

Basic ResearchBiology
Materials and Methods
Cell Cultures The DPSCs (10) and PLSCs (12) were donated under a material transfer agreement with the National Institutes of Dental and Craniofacial Research (Bethesda, MD). Rat fibroblast L929 cells (ATTC, Manassas, VA) were used as a control treatment group cell line. The cells were cultured in Dulbeccos modified Eagles medium (DMEM; BD Biosciences, Franklin Lakes, NJ) supplemented with 10% heat-inactivated fetal calf serum (HyClone, Logan, UT). Cell cultures were maintained at 37C in a humidified atmosphere of 5% CO2 with the culture media being replenished every second day for up to 14 days. Confluent cell cultures were collected by trypsinization (0.25% trypsin/EDTA; Mediatech, Inc, Herndon, VA). Preparation of Three-dimensional Scaffolds for Cell Culture Three types of three-dimensional scaffolds were investigated: (1) open polylactic acid (polymer) scaffolds, (2) bovine collagen (collagen) scaffolds, and (3) calcium phosphate bioceramic (calcium phosphate) scaffolds. All were supplied by BD Biosciences (Bedford, MA). Each scaffold was soaked in neutral phosphate-buffered saline (PBS) and stored at 4C. Before use, the PBS was replaced by culture medium. The scaffolds were incubated in DMEM at 37C for 30 minutes before application of the cells to equalize culture conditions between the scaffolds and cells. Creation of Tissue Constructs The DPSCs, PLSCs, and L929s were added to the scaffolds in fresh aliquots with the aid of a sterile microsyringe; each scaffold was seeded with a million (106) cells (23). The scaffolds were maintained in 24-well culture plates (BD Biosciences) containing 1 mL of culture media. The DMEM culture media was removed and replenished every 2 days (23). During culture, 0.0016% neutral red dye (J.T. Baker, Phillipsburg, NJ) cell viability marker (27) was added to the DMEM in order to stain the vital cells dark red. The DPSCs, PLSCs, and L929s were cultured for 3, 7, and 14 days on the polymer, collagen, and calcium phosphate scaffolds. Each of the scaffold/cell/time tissue construct treatments was replicated four times, providing a total of 144 samples. Histology of Tissue Constructs The tissue constructs were removed from cell culture and fixed by submerging them in a 10% neutral-buffered formalin solution at 18C for 24 hours. The polymer and calcium phosphate tissue constructs were demineralized in 10% formic acid (VWR Suwanne, GA) for 7 days. All the tissue constructs were then dehydrated in a graded series of alcohols from 70% to 100% and then submerged in histoclear (VWR Suwanne, GA) for 5 hours. The constructs were then embedded in paraffin wax blocks and cut into serial histologic sections of 5-m thickness using a microtome. The histology sections were then mounted onto glass slides and covered with a cover slip using adhesive. Pathohistometric Analysis of Tissue Constructs The numbers of stained neutral red cells were counted as the number of vital metabolically active cells (27) within each of the tissue constructs. The cells were counted using pathohistometric analysis, the numbers of cells per microscope field with 5 random microscope fields being counted per specimen using a light microscope at 200 magnification with a reticule (28). The location of the cells within the tissue construct was determined as (1) peripherally distributed, (2) evenly distributed, or (3) centrally distributed.
64
Gebhardt et al.

Statistical Analysis The numbers of cells were counted using pathohistometric analysis (28), and their location within the tissue constructs was analyzed by using analysis of variance statistical tests. Scheffes post hoc tests were used to compare differences between the type of scaffold, the type of stem cell, and the amount of time. The raw data were evaluated using STATview software (SAS Inc, Gary, NC) at a confidence level of 95%.

Results
The fewest numbers of surviving DPSCs were observed in calcium phosphate constructs seeded with DPSCs (Fig. 1A) and PLSCs (Fig. 1B). Much greater numbers of surviving DPSCs (Fig. 1C) and PLSCs (Fig. 1D) were observed in polymer constructs, similar to the numbers of surviving DPSCs (Fig. 1E) and PLSCs (Fig. 1F) in the collagen constructs. The pathohistometric analysis revealed that the type of scaffold material used to create the tissue constructs influenced the survival of the DPSCs (p 0.0001) and PLSCs (p 0.0001). However, few differences were observed between cell survival according to cell type (p 0.7496). The numbers of surviving cells within the constructs increased as the length of cell culture time increased (Fig. 2). Slightly greater numbers of DPSCs and PLSCs were observed in the polymer constructs (Fig. 2). There was no major difference between the numbers of surviving cells in the polymer and collagen tissue constructs at 14 days (p 0.1453) (Fig. 2). After 3 days of cell culture, very few L929 cells attached within the tissue constructs, but the numbers of cells increased over time to a level similar to DPSCs and PLSCs (Fig. 3). There appeared to be few differences among the DPSCs, PLSCs, and L929s according to their ability to survive within tissue constructs (Fig. 3). Calcium phosphate constructs had the highest occurrence of single cells or no cells, whereas, among the polymer constructs, there were zero samples that contained single cells or where no cells were observed. Only one collagen construct appeared to contain mainly single cells; all of the other constructs contained mainly proliferating cell colonies. Very few cells were observed only peripherally or centrally within the constructs; the majority

Figure 1. Surviving DPSCs within the tissue constructs after 14 days.

JOE Volume 35, Number 1, January 2009

Basic ResearchBiology
of the constructs had cells that were evenly distributed, and there were few differences between the scaffold types (p 0.4492).

Discussion
The isolation and availability of dental stem cells, combined with technological advances that allow the fabrication of inexpensive scaffold biomaterials, have now reached the stage of development in which it is possible to create de novo dental tissue constructs in the laboratory. The present study provides further proof of concept of how future regenerative dental treatments may develop, which would involve the transplantation of tissue constructs in the laboratory to replace and regenerate diseased, traumatized, or missing tissues (9). Only recently has this type of study become technically possible because the dental stem cells were first isolated in 2002 (10), and suitable inexpensive scaffold materials have only just become commercially available. The creation of tissue constructs requires a three-dimensional scaffold or membrane to accomplish a biomimetic architecture that maintains cell survival and function (29). In the present study, DPSCs and PLSCs were selected because of their potential to be used as part of dental treatments. The polymer, collagen, and calcium phosphate scaffolds were selected because they represent the three most common types of scaffolds that are used in tissue-engineering research (14 26). The various combinations of dental stem cells and scaffold materials were evaluated to determine the most optimal protocol to create de novo dental tissue constructs. Both the polymer and collagen scaffold constructs had significantly more cell survival than the calcium phosphate scaffold constructs (Fig. 2). These results suggest that the characteristics of the polymer and collagen scaffolds are biocompatible and suitable for maintaining the survival of DPSCs and PLSCs. The numbers of surviving dental stem cells within the scaffolds increased over time (Fig. 3), suggesting that prolonged maintenance in cell culture may be beneficial for more advanced tissue formation and development. In the present study, the constructs were maintained in cell culture for up to 14 days, which revealed that polymer and collagen constructs but not calcium phosphate constructs, may have the potential for use in regenerative endodontics and periodontics. The calcium phosphate scaffold has been used successfully to maintain the osteogenic differentiation of human bone marrow stromal cells (25). However, it is not clear why this scaffold was not successful in supporting the survival of the DPSCs and PLSCs. Some reasons for the lack of biocompatibility of the calcium phosphate scaffolds are that they have the wrong pH, surface characteristics, or formulation to maintain DPSC and PLSC survival. Further research is needed to define the optimal characteristics of scaffold

Figure 3. A bar chart of the mean cell proliferation within the tissue constructs.

materials to be used to maintain DPSC and PLSC survival to create dental tissue constructs. The de novo creation of dental tissue constructs in the laboratory followed by their implantation into a root canal has a potential advantage over the equivalent treatment; that requires host cells from a blood clot to accomplish dental pulp regeneration (6 8). This is because the tissue construct already has a preformed replacement tissue structure, whereas the stem cells within the blood clot need months to form a functional replacement dental tissue (4 8). Both the blood clot and the implantation of a tissue construct will depend on revascularization to deliver nutrients and to remove metabolic waste products from the regenerated tissues. It has been 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 (30, 31). Preclinical and clinical research is needed to directly compare the effectiveness of implanted dental tissue constructs versus blood clots to revitalize teeth. Additional characterizations of the scaffolds, including in vivo implantation and analyses of mineralized tissue formation over a 20- to 25-week period, may be required to more fully assess the utility of each type of scaffold. The clinical introduction of stem cell regenerative endodontic and periodontal treatments can only be delivered to patients after federal efficacy and safety assurance requirements have been addressed. Once approved, the introduction of regenerative endodontic treatments has the potential to benefit hundreds of millions of dental patients each year. The benefits of regenerative endodontics include the ability to continue root development in immature teeth and to revitalize diseased teeth, which may restore their ability to heal in response to disease and trauma. The ideal design of the dental pulp constructs is to be the same shape as gutta-percha cones to accomplish a good fit when inserted into the root canal. The ideal design of the periodontal constructs is to be the same shape as general periodontal barrier membranes, and its benefits include the replacement of diseased and traumatized periodontal tissues.

References
1. Schallhorn RG. The use of autogenous hip marrow biopsy implants for bony crater defects. J Periodontol 1968;39:1457. 2. Schallhorn RG, Hiatt WH, Boyce W. Iliac transplants in periodontal therapy. J Periodontol 1970;41:566 80. 3. Rummelhart JM, Mellonig JT, Gray JL, Towle HJ. A comparison of freeze-dried bone allograft and demineralized freeze-dried bone allograft in juman periodontal osseous defects. J Periodontol 1989;60:655 63. 4. Nygaard-stby B, Hjortdal O. Tissue formation in the root canal following pulp removal. Scand J Dent Res 1971;79:333 49.

Figure 2. A bar chart of the mean numbers of dental stem cells within the tissue constructs.

JOE Volume 35, Number 1, January 2009

Cell Survival within Periodontal Constructs

65

Basic ResearchBiology
5. Hrsted P, Nygaard-stby B. Tissue formation in the root canal after total pulpectomy and partial root filling. Oral Surg Oral Med Oral Pathol 1978;46:275 82. 6. Banchs F, Trope M. Revascularization of immature permanent teeth with apical periodontitis: new treatment protocol? J Endod 2004;30:196 200. 7. Rule DC, Winter GB. Root growth and apical repair subsequent to pulpal necrosis in children. Br Dent J 1966;120:586 90. 8. Iwaya S, Ikawa M, Kubota M. Revascularization of an immature permanent tooth with apical periodontitis and sinus tract. Dent Traumatol 2001;17:1857. 9. Murray PE, Garcia-Godoy F, Hargreaves KM. Regenerative endodontics: a review of current status and a call for action. J Endod 2007;33:37790. 10. Gronthos S, Brahim J, Li W, et al. Stem cell properties of human dental pulp stem cells. J Dent Res 2002;81:5315. 11. Miura M, Gronthos S, Zhao M, et al. SHED: stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci U S A 2003;100:580712. 12. Seo BM, Miura M, Sonoyama W, Coppe C, Stanyon R, Shi S. Recovery of stem cells from cryopreserved periodontal ligament. J Dent Res 2005;84:90712. 13. Gomillion CT, Burg KJ. Stem cells and adipose tissue engineering. Biomaterials 2006; 27:6052 63. 14. Ratanavaraporn J, Kanokpanont S, Tabata Y, Damrongsakkul S. Effects of acid type on physical and biological properties of collagen scaffolds. J Biomater Sci Polym Ed 2008;19:94552. 15. Cooper JA Jr, Sahota JS, Gorum WJ 2nd, Carter J, Doty SB, Laurencin CT. Biomimetic tissue-engineered anterior cruciate ligament replacement. Proc Natl Acad Sci U S A 2007;104:3049 54. 16. Prescott RS, Alsanea R, Fayad MI, et al. In vivo generation of dental pulp-like tissue by using dental pulp stem cells, a collagen scaffold, and dentin matrix protein 1 after subcutaneous transplantation in mice. J Endod 2008;34:421 6. 17. Zhang W, Walboomers XF, van Kuppevelt TH, Daamen WF, Bian Z, Jansen JA. The performance of human dental pulp stem cells on different three-dimensional scaffold materials. Biomaterials 2006;27:5658 68. 18. Seunarine K, Gadegaard N, Tormen M, Meredith DO, Riehle MO, Wilkinson CD. 3D polymer scaffolds for tissue engineering. Nanomed 2006;1:28196. 19. Brauer DS, Rssel C, Vogt S, Weisser J, Schnabelrauch M. Fabrication and in vitro characterization of porous biodegradable composites based on phosphate glasses 20. and oligolactide-containing polymer networks. J Biomed Mater Res A 2007;80: 410 20. Young CS, Terada S, Vacanti JP, Honda M, Bartlett JD, Yelick PC. Tissue engineering of complex tooth structures on biodegradable polymer scaffolds. J Dent Res 2002;81:695700. Duailibi MT, Duailibi SE, Young CS, Bartlett JD, Vacanti JP, Yelick PC. Bioengineered teeth from cultured rat tooth bud cells. J Dent Res 2004;83:523 8. Wang Y, Xia H, Zhao Y, Jiang T. Three-dimensional culture of human periodontal ligament cells on highly porous polyglycolic acid scaffolds in vitro. Conf Proc IEEE Eng Med Biol Soc 2005;5:4908 11. Gotlieb EL, Murray PE, Namerow KN, Kuttler S, Garcia-Godoy F. An ultrastructural investigation of tissue-engineered pulp constructs implanted within endodontically treated teeth. J Am Dent Assoc 2008;139:457 65. Enkel B, Dupas C, Armengol V, et al. Bioactive materials in endodontics. Expert Rev Med Devices 2008;5:47594. Liu G, Zhao L, Cui L, Liu W, Cao Y. Tissue-engineered bone formation using human bone marrow stromal cells and novel beta-tricalcium phosphate. Biomed Mater. 2007;2:78 86. Meek MF, Coert JH. US Food and Drug Administration/Conformit Europe-approved absorbable nerve conduits for clinical repair of peripheral and cranial nerves. Ann Plast Surg 2008;60:110 6. Patel S, Dumsha TC, Sydiskis RJ. Determining periodontal ligament (PDL) cell vitality from exarticulated teeth stored in saline or milk using fluorescein diacetate. Int Endod J 1994;27:15. Murray PE, Smith AJ, Garcia-Godoy F, Lumley PJ. Comparison of operative procedure variables on pulpal viability in an ex vivo model. Int Endod J 2008;41:389 400. Moutos FT, Freed LE, Guilak F. A biomimetic three-dimensional woven composite scaffold for functional tissue engineering of cartilage. Nat Mater 2007;6:1627. Banchs F, Trope M. Revascularization of immature permanent teeth with apical periodontitis: new treatment protocol? J Endod. 2004;30:196 200. Chueh LH, Huang GT. Immature teeth with periradicular periodontitis or abscess undergoing apexogenesis: a paradigm shift. J Endod 2006;32:120513.

21. 22.

23.

24. 25.

26.

27.

28. 29.

30. 31.

66

Gebhardt et al.

JOE Volume 35, Number 1, January 2009