- Email: [email protected]
Effects of Cryopreservation of Intact Teeth on the Isolated Dental Pulp Stem Cells
Basic Research—Biology
Effects of Cryopreservation of Intact Teeth on the Isolated Dental Pulp Stem Cells Sheng-Yang Lee, DDS, PhD,* Pao-Chang Chiang, DDS, MS,* Yu-Hui Tsai, PhD,† Shih-Ying Tsai, PhD,† Jiiang-Huei Jeng, DDS, PhD,‡ Toshitsugu Kawata, DDS, PhD,§ and Haw-Ming Huang, PhDk Abstract Introduction: Human dental pulp stem cells (DPSCs) have been reported to be useful material for future regenerative medicine. Clinically, cryopreservation of intact teeth can successfully preserve the periodontal ligament for future autotransplantation; however, the effects of cryopreservation procedure on the properties of DPSCs are still unclear. The aim of this study was to test whether DPSCs isolated from cryopreserved teeth can express stem cell–specific markers. Methods: In this study, a novel programmable freezer coupled to a magnetic field was used to perform the cryopreservation experiments. The tested DPSCs were isolated from magnetically cryopreserved and non-cryopreserved fresh teeth with an enzyme digestion procedure. The success rate of isolation, growth curves, morphology, stem cell–specific markers, and the differentiation capacity of the isolated cells were evaluated and compared. Results: The isolation rate of dental pulp cells from magnetically cryopreserved teeth was 73%. After culture for 5 generations, there was no significant difference in cell viability between cells isolated from magnetically cryopreserved teeth and those isolated from fresh teeth. There were also no visible differences between the 2 groups of dental pulp cells in morphology, expression of stem cell markers, or osteogenic and adipogenic differentiations. Conclusions: The results suggest that cryopreserved whole teeth can be used for autotransplantation and provide a viable source of DPSCs. (J Endod 2010;36:1336–1340)
S
Key Words
Cryopreservation and Cell Culture Normal human premolars were collected from adults (aged 18–30 years) at the Department of Orthodontics, Wan-Fang Medical Center, Taipei Medical University, Taipei, Taiwan. Immediately after extraction, teeth were cleaned with phosphate-buffered saline and stored in cryoprotectant (BAMBANKER, Lymphotec, Tokyo, Japan). The teeth were divided into 2 groups. Teeth in the magnetically cryopreserved group were cryopreserved in a program freezer (ABI, Chiba, Japan) supplied with a slight magnetic field. Briefly, the teeth were transported at 4 C and then placed in a freezer at –5 C. Teeth were maintained at that temperature for 15 minutes and then cooled at a rate of –0.5 C/min until the temperature reached –32 C. After the freezing procedure, the experimental teeth were transferred to a freezer (MDF-11561; Sanyo, Osaka, Japan) and stored at –152 C for 7 days. The time period was chosen according to a previous report that demonstrated no significant difference in the amount of revascularization between teeth stored in a tooth bank for 7 days and those immediately transplanted without freezing (10). The control group was composed of fresh teeth that had been extracted from the contralateral side of each patient. Those teeth were not subjected to the cryopreservation procedure. Biologic tests performed on the control teeth were done immediately after extraction and cleaning.
Cryopreservation, dental pulp stem cells, differentiation
From the *School of Dentistry, †Graduate Institute of Medical Sciences, and kGraduate Institute of Biomedical Materials and Engineering, Taipei Medical University, Taipei, Taiwan; ‡School of Dentistry, National Taiwan University, Taipei, Taiwan; and § Department of Orthodontics, Hiroshima University, Hiroshima, Japan. Drs Lee and Chiang contributed equally to this work. Address requests for reprints to Dr Haw-Ming Huang, Graduate Institute of Biomedical Materials & Engineering, Taipei Medical University, 250 Wu-Hsing Street, Taipei, Taiwan. E-mail address: [email protected]. 0099-2399/$0 - see front matter Copyright ª 2010 American Association of Endodontists. doi:10.1016/j.joen.2010.04.015
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Lee et al.
everal studies have indicated that postnatal stem cells are present in bone marrow, neural tissue, skin, and retina (1). These cells exhibit capacities for differentiation into various cells and development into diverse tissue. These self-renewal capabilities make stem cells become an effective material for regenerative medicine. Human dental pulp stem cells (DPSCs) were first reported in 2000 (2). Unlike bone marrow–derived stem cells, DPSCs can be isolated with noninvasive procedures. In addition, unlike the use of embryonic stem cells, the use of DPSCs in research and therapy is not considered to be controversial (3). In 2007, Jo et al (1) isolated postnatal stem cells from human dental tissues such as dental pulp, periodontal ligament, periapical follicle, and the surrounding mandibular bone marrow and found that those stem cells were able to differentiate into osteoblasts and adipocytes. Because of their multipotent differentiation ability and immunosuppressive activity, dental stem cells provide an alternative to stem cells from other sources for use in regenerative medicine (4, 5). It is believed that DPSCs will play an important role in regenerative endodontics in the near future (6). Although isolation of DPSCs from fresh teeth is possible (7), contamination and damage during long-term cryostorage might have an effect on the viability of dental pulp stem cells. Cryopreservation on various tissue and cells has been investigated for several decades and has become an important issue for tissue engineering (8, 9). Laureys et al (10) cryopreserved pulpless teeth in liquid nitrogen at –196 for 7 days. Although their experiments demonstrated that pulpless teeth can revascularize after being cryopreserved in a tooth bank for 1 week, DPSC viability and functions were not assessed (10). Recently, recovery of DPSCs from cryopreserved intact teeth was achieved by Woods et al (11). However, stem cell–specific markers CD-44 and STRO-1 were not examined in their study. The aim of this study was to confirm whether DPSCs isolated from cryopreserved teeth would survive and function normally after thawing.
Materials and Methods
JOE — Volume 36, Number 8, August 2010
Basic Research—Biology
Figure 1. (A) and (B) are inverted microscope images of pulp cells from fresh and cryopreserved teeth, respectively. Cells with fibroblast-like morphology can be found in both groups. (C) Cell growth viability was assessed by the MTT method. The growth curve shows no statistical difference between fresh and magnetically cryopreserved teeth. (D) One example of flow cytometry histogram demonstrated STRO-1 expression of DPSCs isolated from cryopreservation.
After 7 days of cryopreservation, the teeth were thawed, and dental pulp cells were isolated with a modified enzyme digestion method (2). Briefly, minced pulp tissue was digested in an enzyme mixture of 4 mg/ mL collagenase type I (Sigma, St Louis, MO) and 2 mg/mL dispase (Sigma) in a 37 C water bath. Cultures were then incubated at 37 C in a humidified atmosphere of 95% air and 5% CO2. To assess the success rate of culturing pulp cells after cryopreservation, the cell numbers on the 30th day after primary culture were counted. In this study, we defined the average cell number counted from non-cryopreserved teeth on the 30th day as the threshold (6 104 cells/mL). A successful culture was defined as one in which the cell numbers on day 30 were larger than that threshold. In this experiment, at least 8 samples from each experimental group were used. All experimental protocols were approved by the Committee on Human Research, Taipei Medical University. This information was also provided to the patients whose teeth were collected, and an agreement was signed by patients before the experiment.
Cell Viability and Morphology Examination In the following experiments, 5–10 passages of cultured dental pulp cells were used. Viability of DPSCs in the magnetically cryopreserved and non-cryopreserved groups was evaluated by a modified 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyl tetrazolium (MTT) (Sigma) assay. After incubation for 24, 48, 72, 96, and 120 hours, MTT working solution was added. The formazan salt was lysed with dimethyl JOE — Volume 36, Number 8, August 2010
sulfoxide, and the absorbance at 570 nm/690 nm was measured (n = 4). Student t test was used to analyze the significance between the 2 groups. The level of significance was set at .05. Morphologic changes in the cultured cells were assessed when the cells were incubated for 3 days by using an inverted microscope. For each sample, 9 random fields within a sample were examined. To confirm the DPSC population in the cultured dental pulp cells, STRO-1 marker was examined by flow cytometry. Cultured pulp cells of both cryopreserved and non-cryopreserved groups were stained with fluorescein isothiocyanate (FITC)–conjugated antibody against STRO-1 (sc-47733; Santa Cruz Biotechnology Inc, Santa Cruz, CA) and were analyzed by using a FACSCalibur instrument and CellQuest software (Becton-Dickson, Franklin Lakes, NJ).
Examination of Stem Cell–specific Markers For CD-34 examination, mouse anti-human CD-34 monoclonal immunoglobulin G (sc-7324; Santa Cruz Biotechnology Inc) and FITC-conjugated goat anti-mouse immunoglobulin G (H+L; Jackson Immuno Research Laboratories Inc, West Grove, PA) were used as primary and secondary antibodies, respectively. For CD-44 and STRO-1 double staining, mouse monoclonal antibody against CD-44 (sc-7297; Santa Cruz Biotechnology Inc) and FITC-conjugated antibody against STRO-1 (sc-47733; Santa Cruz Biotechnology Inc) were used. After 48 hours of incubation, the cells of cryopreserved and noncryopreserved groups were fixed in 4% paraformaldehyde for 20 Effects of Cryopreservation of Intact Teeth on Isolated DPSCs
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Basic Research—Biology
Figure 2. Double stains of the DPSCs isolated from fresh (A) and magnetically cryopreserved (D) teeth. Expression of CD44 (B, E) and STRO-1 (C, F) was found in the fresh and magnetically cryopreserved teeth. Expressions of CD44 and STRO-1 were stained as red and green, respectively. (This figure is available in color online at www.aae.org/joe/.)
Results
ervation. Cells from fresh and cryopreserved teeth showed fibroblastlike morphology. There were no visible differences in morphology between the 2 groups (Fig. 1A, B). Fig. 1C demonstrates the growth curves of the cultured pulp cells. After culturing for 5 days, the viability of cells isolated from magnetically cryopreserved teeth had increased by 3.15 times, and that of cells from fresh teeth had increased by 3.31 times. There was no significant difference in cell viability between the 2 groups. Flow cytometry tests showed that the fluorescent intensity of STRO-1 stained dental pulp cells cultured from fresh and cryopreserved teeth is slightly larger than control. Quantified analysis indicated that the population of DPSCs in the cultured pulp cells was 9% (Fig. 1D). Immunostaining of CD34, CD44, and STRO-1 was performed to identify the dental pulp stem cells. Cells from both groups showed negative expression for CD34 (data not shown) but positive expression for CD44 and STRO-1 (Fig. 2). To test the multi-differentiation ability of the cryopreserved DPSCs, adipogenic and osteogenic differentiations were tested. As shown in Fig. 3, DPSCs from both groups were able to differentiate into adipocytes and osteocytes. Adipogenesis was confirmed by the presence of fat droplets (Fig. 3C, D), and osteogenesis was confirmed by the presence of calcium deposition (Fig. 3G, H). Neither adipogenesis (Fig. 3A, B) nor osteogenesis (Fig. 3E, F) was found in the noninduced cells.
We found that the culture rate of isolated pulp cells from fresh teeth was 100%, and the culture rate of cells from cryopreserved teeth was 73%. Images taken with an inverted microscope were used to observe the changes in morphology of the cultured cells after cryopres-
The aim of this study was to evaluate whether DPSCs could be preserved and then isolated from teeth that had been subjected to
minutes and then incubated overnight with primary antibodies (1:500 dilutions). The samples were subsequently incubated with secondary antibodies for 1 hour at 37 C. Finally, the samples were examined by confocal microscopy (TCS SP5; Leica Microsystems CMS GmbH, Mannheim, Germany).
Ability of the Multi-lineage Differentiation The medium of cultured cells was changed 2 times a week until confluence was achieved. Then the medium was replaced by differentiation-inducing medium. The differentiation-inducing medium was supplemented with 0.5 mmol/L isobutylmethylxanthine, 60 mmol/L indomethacin, 0.5 mmol/L hydrocortisone, and 10 mg mL insulin to induce adipogenesis and was supplemented with 0.01 mmol/L dexamethasone, 1.8 mmol/L KH2PO4 to induce osteogenesis. The samples were then incubated for another 28 days, with 2 medium changes per week. As a control, the noninduced cells were incubated with inducing chemical-free medium. At the end of the cultivation period, the cells were fixed with 4% formaldehyde and then stained with oil-red-O and alizarin-red-S to stain lipid droplets and calcium deposition, respectively.
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Discussion
JOE — Volume 36, Number 8, August 2010
Basic Research—Biology
Figure 3. DPSCs from fresh (C, G) teeth and those from magnetically cryopreserved (D, H) teeth show the ability to differentiate into adipocytes and osteocytes. Adipogenesis (C, D) was confirmed by the presence of fat droplets (black arrow), and osteogenesis (G, H) was confirmed by the presence of calcium deposition (red color). (This figure is available in color online at www.aae.org/joe/.)
a cryopreservation process. Our results showed that the rate of cell culture from cryopreserved teeth was 73%. In addition, the cells isolated from cryopreserved teeth not only maintained their growth potential but also demonstrated a high efficiency in osteogenic and adipodenic differentiations (Fig. 3D, H). JOE — Volume 36, Number 8, August 2010
In previous studies, the morphology of DPSCs was described as being similar to that of fibroblast-like cells (2) or bone marrow stem cells with spindle shape (12). In this study, the morphology data showed similar variety of cells in both groups (Fig. 1A, B). Magnetic cryopreservation in this study had no effect on the morphology of the
Effects of Cryopreservation of Intact Teeth on Isolated DPSCs
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Basic Research—Biology isolated cells. Postnatal adult stem cells were reported to have great therapeutic potential because of their self-renewal and their potential to differentiate into multiple cell lineages (3), including odontoblasts (13), adipocytes, chondrocytes (14), osteocytes (3), and neuronlike cells (15). However, none of the cells from either group expressed the neuron cell–specific markers GAP43 and CRMP-2 (data not shown). According to Shi et al (16), DPSCs do not express the hematopoietic stem cell marker CD34 but do express CD44 and STRO-1. We found similar findings in our study, in which DPSCs isolated from magnetically cryopreserved and those from fresh teeth were negative for CD34 but positive for CD44 (Fig. 2B, E) and STRO-1 (Fig. 2C, F). Those data suggest that the surface markers of DPSCs are not influenced by the cryopreservation procedures used in this study. The results of this study indicate that dental pulp stem cells isolated from cryopreserved teeth maintain their growth potential, surface markers, and, most importantly, their osteogenic and adipogenic differentiation ability. Although a previous report indicated that teeth can revascularize after autotransplantation only when the original tissue is removed at the moment of extraction (10), we found that DPSCs can be isolated from a preserved state after thawing. These results could be a useful reference for expanding the applications of tooth banking from cryopreservation for autotransplantation to storage of DPSCs.
Acknowledgments The authors would like to thank ABI Ltd (Chiba, Japan) and Dr Geroge T.-J Huang (Boston University Henry M. Goldman School of Dental Medicine) for freezing and DPSC isolation technique support, respectively.
References 1. Jo YY, Lee HJ, Kook SY, et al. Isolation and characterization of postnatal stem cells from human dental tissues. Tissue Eng 2007;13:767–73.
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2. Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A 2000;97:13625–30. 3. Huang AH, Snyder BR, Cheng PH, Chan AW. Putative dental pulp-derived stem/ stromal cells promote proliferation and differentiation of endogenous neural cells in the hippocampus of mice. Stem Cells 2008;26:2654–63. 4. Pierdomenico L, Bonsi L, Calvitti M, et al. Multipotent mesenchymal stem cells with immunosuppressive activity can be easily isolated from dental pulp. Transplantation 2005;80:836–42. 5. Gebhardt M, Murray PE, Namerow KN, Kuttler S, Garcia-Godoy F. Cell survival within pulp and periodontal constructs. J Endod 2009;35:63–6. 6. Murray PE, Garcia-Godoy F, Hargreaves KM. Regenerative endodontics: a review of current status and a call for action. J Endod 2007;33:377–90. 7. Zhang W, Walboomers XF, Shi S, Fan M, Jansen JA. Multilineage differentiation potential of stem cells derived from human dental pulp after cryopreservation. Tissue Eng 2006;12:2813–23. 8. Sumida S. Transfusion and transplantation of cryopreserved cells and tissues. Cell Tissue Bank 2006;7:265–305. 9. Andreasen JO, Paulsen HU, Yu Z, Bayer T, Schwartz O. A long-term study of 370 autotransplanted premolars: part II–tooth survival and pulp healing subsequent to transplantation. Eur J Orthod 1990;12:14–24. 10. Laureys W, Beele H, Cornelissen R, Dermaut L. Revascularization after cryopreservation and autotransplantation of immature and mature apicoectomized teeth. Am J Orthod Dentofacial Orthop 2001;119:346–52. 11. Woods EJ, Perry BC, Hockema JJ, Larson L, Zhou D, Goebel WS. Optimized cryopreservation method for human dental pulp-derived stem cells and their tissues of origin for banking and clinical use. Cryobiology 2009;59:150–7. 12. Huang AH, Chen YK, Lin LM, Shieh TY, Chan AW. Isolation and characterization of dental pulp stem cells from a supernumerary tooth. J Oral Pathol Med 2008;37: 571–4. 13. Couble ML, Farges JC, Bleicher F, Perrat-Mabillon B, Boudeulle M, Magloire H. Odontoblast differentiation of human dental pulp cells in explant cultures. Calcif Tissue Int 2000;66:129–38. 14. Kawazoe Y, Katoh S, Onodera Y, Kohgo T, Shindoh M, Shiba T. Activation of the FGF signaling pathway and subsequent induction of mesenchymal stem cell differentiation by inorganic polyphosphate. Int J Biol Sci 2008;4:37–47. 15. Iohara K, Zheng L, Ito M, Tomokiyo A, Matsushita K, Nakashima M. Side population cells isolated from porcine dental pulp tissue with self-renewal and multipotency for dentinogenesis, chondrogenesis, adipogenesis, and neurogenesis. Stem Cells 2006; 24:2493–503. 16. Shi S, Bartold PM, Miura M, Seo BM, Robey PG, Gronthos S. The efficacy of mesenchymal stem cells to regenerate and repair dental structures. Orthod Craniofac Res 2005;8:191–9.
JOE — Volume 36, Number 8, August 2010
Effects of Cryopreservation of Intact Teeth on the Isolated Dental Pulp Stem Cells Sheng-Yang Lee, DDS, PhD,* Pao-Chang Chiang, DDS, MS,* Yu-Hui Tsai, PhD,† Shih-Ying Tsai, PhD,† Jiiang-Huei Jeng, DDS, PhD,‡ Toshitsugu Kawata, DDS, PhD,§ and Haw-Ming Huang, PhDk Abstract Introduction: Human dental pulp stem cells (DPSCs) have been reported to be useful material for future regenerative medicine. Clinically, cryopreservation of intact teeth can successfully preserve the periodontal ligament for future autotransplantation; however, the effects of cryopreservation procedure on the properties of DPSCs are still unclear. The aim of this study was to test whether DPSCs isolated from cryopreserved teeth can express stem cell–specific markers. Methods: In this study, a novel programmable freezer coupled to a magnetic field was used to perform the cryopreservation experiments. The tested DPSCs were isolated from magnetically cryopreserved and non-cryopreserved fresh teeth with an enzyme digestion procedure. The success rate of isolation, growth curves, morphology, stem cell–specific markers, and the differentiation capacity of the isolated cells were evaluated and compared. Results: The isolation rate of dental pulp cells from magnetically cryopreserved teeth was 73%. After culture for 5 generations, there was no significant difference in cell viability between cells isolated from magnetically cryopreserved teeth and those isolated from fresh teeth. There were also no visible differences between the 2 groups of dental pulp cells in morphology, expression of stem cell markers, or osteogenic and adipogenic differentiations. Conclusions: The results suggest that cryopreserved whole teeth can be used for autotransplantation and provide a viable source of DPSCs. (J Endod 2010;36:1336–1340)
S
Key Words
Cryopreservation and Cell Culture Normal human premolars were collected from adults (aged 18–30 years) at the Department of Orthodontics, Wan-Fang Medical Center, Taipei Medical University, Taipei, Taiwan. Immediately after extraction, teeth were cleaned with phosphate-buffered saline and stored in cryoprotectant (BAMBANKER, Lymphotec, Tokyo, Japan). The teeth were divided into 2 groups. Teeth in the magnetically cryopreserved group were cryopreserved in a program freezer (ABI, Chiba, Japan) supplied with a slight magnetic field. Briefly, the teeth were transported at 4 C and then placed in a freezer at –5 C. Teeth were maintained at that temperature for 15 minutes and then cooled at a rate of –0.5 C/min until the temperature reached –32 C. After the freezing procedure, the experimental teeth were transferred to a freezer (MDF-11561; Sanyo, Osaka, Japan) and stored at –152 C for 7 days. The time period was chosen according to a previous report that demonstrated no significant difference in the amount of revascularization between teeth stored in a tooth bank for 7 days and those immediately transplanted without freezing (10). The control group was composed of fresh teeth that had been extracted from the contralateral side of each patient. Those teeth were not subjected to the cryopreservation procedure. Biologic tests performed on the control teeth were done immediately after extraction and cleaning.
Cryopreservation, dental pulp stem cells, differentiation
From the *School of Dentistry, †Graduate Institute of Medical Sciences, and kGraduate Institute of Biomedical Materials and Engineering, Taipei Medical University, Taipei, Taiwan; ‡School of Dentistry, National Taiwan University, Taipei, Taiwan; and § Department of Orthodontics, Hiroshima University, Hiroshima, Japan. Drs Lee and Chiang contributed equally to this work. Address requests for reprints to Dr Haw-Ming Huang, Graduate Institute of Biomedical Materials & Engineering, Taipei Medical University, 250 Wu-Hsing Street, Taipei, Taiwan. E-mail address: [email protected]. 0099-2399/$0 - see front matter Copyright ª 2010 American Association of Endodontists. doi:10.1016/j.joen.2010.04.015
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Lee et al.
everal studies have indicated that postnatal stem cells are present in bone marrow, neural tissue, skin, and retina (1). These cells exhibit capacities for differentiation into various cells and development into diverse tissue. These self-renewal capabilities make stem cells become an effective material for regenerative medicine. Human dental pulp stem cells (DPSCs) were first reported in 2000 (2). Unlike bone marrow–derived stem cells, DPSCs can be isolated with noninvasive procedures. In addition, unlike the use of embryonic stem cells, the use of DPSCs in research and therapy is not considered to be controversial (3). In 2007, Jo et al (1) isolated postnatal stem cells from human dental tissues such as dental pulp, periodontal ligament, periapical follicle, and the surrounding mandibular bone marrow and found that those stem cells were able to differentiate into osteoblasts and adipocytes. Because of their multipotent differentiation ability and immunosuppressive activity, dental stem cells provide an alternative to stem cells from other sources for use in regenerative medicine (4, 5). It is believed that DPSCs will play an important role in regenerative endodontics in the near future (6). Although isolation of DPSCs from fresh teeth is possible (7), contamination and damage during long-term cryostorage might have an effect on the viability of dental pulp stem cells. Cryopreservation on various tissue and cells has been investigated for several decades and has become an important issue for tissue engineering (8, 9). Laureys et al (10) cryopreserved pulpless teeth in liquid nitrogen at –196 for 7 days. Although their experiments demonstrated that pulpless teeth can revascularize after being cryopreserved in a tooth bank for 1 week, DPSC viability and functions were not assessed (10). Recently, recovery of DPSCs from cryopreserved intact teeth was achieved by Woods et al (11). However, stem cell–specific markers CD-44 and STRO-1 were not examined in their study. The aim of this study was to confirm whether DPSCs isolated from cryopreserved teeth would survive and function normally after thawing.
Materials and Methods
JOE — Volume 36, Number 8, August 2010
Basic Research—Biology
Figure 1. (A) and (B) are inverted microscope images of pulp cells from fresh and cryopreserved teeth, respectively. Cells with fibroblast-like morphology can be found in both groups. (C) Cell growth viability was assessed by the MTT method. The growth curve shows no statistical difference between fresh and magnetically cryopreserved teeth. (D) One example of flow cytometry histogram demonstrated STRO-1 expression of DPSCs isolated from cryopreservation.
After 7 days of cryopreservation, the teeth were thawed, and dental pulp cells were isolated with a modified enzyme digestion method (2). Briefly, minced pulp tissue was digested in an enzyme mixture of 4 mg/ mL collagenase type I (Sigma, St Louis, MO) and 2 mg/mL dispase (Sigma) in a 37 C water bath. Cultures were then incubated at 37 C in a humidified atmosphere of 95% air and 5% CO2. To assess the success rate of culturing pulp cells after cryopreservation, the cell numbers on the 30th day after primary culture were counted. In this study, we defined the average cell number counted from non-cryopreserved teeth on the 30th day as the threshold (6 104 cells/mL). A successful culture was defined as one in which the cell numbers on day 30 were larger than that threshold. In this experiment, at least 8 samples from each experimental group were used. All experimental protocols were approved by the Committee on Human Research, Taipei Medical University. This information was also provided to the patients whose teeth were collected, and an agreement was signed by patients before the experiment.
Cell Viability and Morphology Examination In the following experiments, 5–10 passages of cultured dental pulp cells were used. Viability of DPSCs in the magnetically cryopreserved and non-cryopreserved groups was evaluated by a modified 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyl tetrazolium (MTT) (Sigma) assay. After incubation for 24, 48, 72, 96, and 120 hours, MTT working solution was added. The formazan salt was lysed with dimethyl JOE — Volume 36, Number 8, August 2010
sulfoxide, and the absorbance at 570 nm/690 nm was measured (n = 4). Student t test was used to analyze the significance between the 2 groups. The level of significance was set at .05. Morphologic changes in the cultured cells were assessed when the cells were incubated for 3 days by using an inverted microscope. For each sample, 9 random fields within a sample were examined. To confirm the DPSC population in the cultured dental pulp cells, STRO-1 marker was examined by flow cytometry. Cultured pulp cells of both cryopreserved and non-cryopreserved groups were stained with fluorescein isothiocyanate (FITC)–conjugated antibody against STRO-1 (sc-47733; Santa Cruz Biotechnology Inc, Santa Cruz, CA) and were analyzed by using a FACSCalibur instrument and CellQuest software (Becton-Dickson, Franklin Lakes, NJ).
Examination of Stem Cell–specific Markers For CD-34 examination, mouse anti-human CD-34 monoclonal immunoglobulin G (sc-7324; Santa Cruz Biotechnology Inc) and FITC-conjugated goat anti-mouse immunoglobulin G (H+L; Jackson Immuno Research Laboratories Inc, West Grove, PA) were used as primary and secondary antibodies, respectively. For CD-44 and STRO-1 double staining, mouse monoclonal antibody against CD-44 (sc-7297; Santa Cruz Biotechnology Inc) and FITC-conjugated antibody against STRO-1 (sc-47733; Santa Cruz Biotechnology Inc) were used. After 48 hours of incubation, the cells of cryopreserved and noncryopreserved groups were fixed in 4% paraformaldehyde for 20 Effects of Cryopreservation of Intact Teeth on Isolated DPSCs
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Basic Research—Biology
Figure 2. Double stains of the DPSCs isolated from fresh (A) and magnetically cryopreserved (D) teeth. Expression of CD44 (B, E) and STRO-1 (C, F) was found in the fresh and magnetically cryopreserved teeth. Expressions of CD44 and STRO-1 were stained as red and green, respectively. (This figure is available in color online at www.aae.org/joe/.)
Results
ervation. Cells from fresh and cryopreserved teeth showed fibroblastlike morphology. There were no visible differences in morphology between the 2 groups (Fig. 1A, B). Fig. 1C demonstrates the growth curves of the cultured pulp cells. After culturing for 5 days, the viability of cells isolated from magnetically cryopreserved teeth had increased by 3.15 times, and that of cells from fresh teeth had increased by 3.31 times. There was no significant difference in cell viability between the 2 groups. Flow cytometry tests showed that the fluorescent intensity of STRO-1 stained dental pulp cells cultured from fresh and cryopreserved teeth is slightly larger than control. Quantified analysis indicated that the population of DPSCs in the cultured pulp cells was 9% (Fig. 1D). Immunostaining of CD34, CD44, and STRO-1 was performed to identify the dental pulp stem cells. Cells from both groups showed negative expression for CD34 (data not shown) but positive expression for CD44 and STRO-1 (Fig. 2). To test the multi-differentiation ability of the cryopreserved DPSCs, adipogenic and osteogenic differentiations were tested. As shown in Fig. 3, DPSCs from both groups were able to differentiate into adipocytes and osteocytes. Adipogenesis was confirmed by the presence of fat droplets (Fig. 3C, D), and osteogenesis was confirmed by the presence of calcium deposition (Fig. 3G, H). Neither adipogenesis (Fig. 3A, B) nor osteogenesis (Fig. 3E, F) was found in the noninduced cells.
We found that the culture rate of isolated pulp cells from fresh teeth was 100%, and the culture rate of cells from cryopreserved teeth was 73%. Images taken with an inverted microscope were used to observe the changes in morphology of the cultured cells after cryopres-
The aim of this study was to evaluate whether DPSCs could be preserved and then isolated from teeth that had been subjected to
minutes and then incubated overnight with primary antibodies (1:500 dilutions). The samples were subsequently incubated with secondary antibodies for 1 hour at 37 C. Finally, the samples were examined by confocal microscopy (TCS SP5; Leica Microsystems CMS GmbH, Mannheim, Germany).
Ability of the Multi-lineage Differentiation The medium of cultured cells was changed 2 times a week until confluence was achieved. Then the medium was replaced by differentiation-inducing medium. The differentiation-inducing medium was supplemented with 0.5 mmol/L isobutylmethylxanthine, 60 mmol/L indomethacin, 0.5 mmol/L hydrocortisone, and 10 mg mL insulin to induce adipogenesis and was supplemented with 0.01 mmol/L dexamethasone, 1.8 mmol/L KH2PO4 to induce osteogenesis. The samples were then incubated for another 28 days, with 2 medium changes per week. As a control, the noninduced cells were incubated with inducing chemical-free medium. At the end of the cultivation period, the cells were fixed with 4% formaldehyde and then stained with oil-red-O and alizarin-red-S to stain lipid droplets and calcium deposition, respectively.
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Discussion
JOE — Volume 36, Number 8, August 2010
Basic Research—Biology
Figure 3. DPSCs from fresh (C, G) teeth and those from magnetically cryopreserved (D, H) teeth show the ability to differentiate into adipocytes and osteocytes. Adipogenesis (C, D) was confirmed by the presence of fat droplets (black arrow), and osteogenesis (G, H) was confirmed by the presence of calcium deposition (red color). (This figure is available in color online at www.aae.org/joe/.)
a cryopreservation process. Our results showed that the rate of cell culture from cryopreserved teeth was 73%. In addition, the cells isolated from cryopreserved teeth not only maintained their growth potential but also demonstrated a high efficiency in osteogenic and adipodenic differentiations (Fig. 3D, H). JOE — Volume 36, Number 8, August 2010
In previous studies, the morphology of DPSCs was described as being similar to that of fibroblast-like cells (2) or bone marrow stem cells with spindle shape (12). In this study, the morphology data showed similar variety of cells in both groups (Fig. 1A, B). Magnetic cryopreservation in this study had no effect on the morphology of the
Effects of Cryopreservation of Intact Teeth on Isolated DPSCs
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Basic Research—Biology isolated cells. Postnatal adult stem cells were reported to have great therapeutic potential because of their self-renewal and their potential to differentiate into multiple cell lineages (3), including odontoblasts (13), adipocytes, chondrocytes (14), osteocytes (3), and neuronlike cells (15). However, none of the cells from either group expressed the neuron cell–specific markers GAP43 and CRMP-2 (data not shown). According to Shi et al (16), DPSCs do not express the hematopoietic stem cell marker CD34 but do express CD44 and STRO-1. We found similar findings in our study, in which DPSCs isolated from magnetically cryopreserved and those from fresh teeth were negative for CD34 but positive for CD44 (Fig. 2B, E) and STRO-1 (Fig. 2C, F). Those data suggest that the surface markers of DPSCs are not influenced by the cryopreservation procedures used in this study. The results of this study indicate that dental pulp stem cells isolated from cryopreserved teeth maintain their growth potential, surface markers, and, most importantly, their osteogenic and adipogenic differentiation ability. Although a previous report indicated that teeth can revascularize after autotransplantation only when the original tissue is removed at the moment of extraction (10), we found that DPSCs can be isolated from a preserved state after thawing. These results could be a useful reference for expanding the applications of tooth banking from cryopreservation for autotransplantation to storage of DPSCs.
Acknowledgments The authors would like to thank ABI Ltd (Chiba, Japan) and Dr Geroge T.-J Huang (Boston University Henry M. Goldman School of Dental Medicine) for freezing and DPSC isolation technique support, respectively.
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JOE — Volume 36, Number 8, August 2010