Cell Transplantation, Vol. 20, pp. 1003 1013, 2011 0963-6897/11 $90.00 +.00 Printed in the USA. All rights reserved. DOI: 10.3727/096368910X539128 Copyright 2011 Cognizant Comm. Corp. E-ISSN 1555-3892 www.cognizantcommunication.com Promising Cell-Based Therapy for Bone Regeneration Using Stem Cells From Deciduous Teeth, Dental Pulp, and Bone Marrow Yoichi Yamada,* Kenji Ito, Sayaka Nakamura, Minoru Ueda, and Tetsuro Nagasaka *Center for Genetic and Regenerative Medicine, Nagoya University School of Medicine, Nagoya, Japan Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine, Nagoya, Japan Laboratory Medicine, Nagoya University Graduate School of Medicine, Nagoya, Japan We attempted to regenerate bone in a significant osseous defect with various stem cells from deciduous teeth, extracted from puppies, and grafted them into a parent canine mandible as an allograft, parent dental pulp, and bone marrow by tissue engineering and regenerative medicine technology using platelet-rich plasma as an autologous scaffold and signal molecules. Initially, teeth were extracted from a child and parent hybrid canine mandible region and bone marrow (canine mesenchymal stem cells; cmscs), and parent teeth (canine dental pulp stem cells; cdpscs), and stem cells were extracted from deciduous teeth (puppy deciduous teeth stem cells; pdtscs). After 4 weeks, bone defects were prepared on both sides of the mandible with a trephine bar. Graft materials were implanted into these defects: 1) control (defect only), 2) plateletrich plasma (PRP), 3) cmscs/prp, 4) cdpscs/prp, and 5) pdtscs/prp to investigate the effect of stem cells. The newly formed bones were evaluated by histology and histomorphometric analysis in the defects at 2, 4, and 8 weeks. According to histological observations, the cmscs/prp, cdpscs/prp, and pdtscs/ PRP groups had well-formed mature bone and neovascularization compared with the control (defect only) and PRP groups at 4 and 8 weeks, respectively, and the mineralized tissues in cmscs/prp, cdpscs/prp, and pdtscs/prp specimens were positive for osteocalcin at 8 weeks. Histometrically, newly formed bone areas were 19.0 ± 2.9% (control), 19.7 ± 6.0% (PRP), 52.8 ± 3.5% (cmscs/prp), 61.6 ± 1.3% (cdpscs/ PRP), and 54.7 ± 2.2% (pdtscs/prp) at 8 weeks. There were significant differences between cmscs, cdpscs, pdtscs/prp, and control and PRP groups. These results demonstrate that stem cells from deciduous teeth, dental pulp, and bone marrow with PRP have the ability to form bone, and bone formation with DTSCs might have the potential to generate a graft between a child and parent. This preclinical study could pave the way for stem cell therapy in orthopedics and oral maxillofacial reconstruction for clinical application. Key words: Tissue engineering and regenerative medicine (TERM); Tissue-engineered bone (TEB); Dental pulp stem cells (DPSCs); Deciduous teeth stem cells (DTSCs); Mesenchymal stem cells (MSCs); Newly formed bone area INTRODUCTION concept is to regenerate tissues using three elements stem cells, scaffolds, and signal molecules and it is thought that the role of stem cells is most important. Recently, embryonic stem cells (ES cells), induced pluripotent stem cells (ips cells), and somatic stem cells have been reported; however, ES and ips cells have many issues to overcome for clinical use because of eth- ical and safety problems, immunorejection, and tumorigenesis. Somatic stem cells, especially mesenchymal stem cells (MSCs) isolated from various tissues, including bone marrow, adipose tissue, skin, umbilical cord, and placenta (7,17,26,28), have been used in clinical applications. We have used bone marrow-derived MSCs (BMMSCs), instead of autogenous bone grafting, in Bone defects often occur from tumor resection, congenital malformation, trauma, osteoporotic fractures, surgery, or periodontitis, and autogenous bone grafting has been the gold standard approach to treatment. However, severe invasiveness with attendant donor site morbidity and complications is not the only issue, as there is also a limited supply of autogenous bone. On the other hand, tissue engineering and regenerative medicine (TERM) using stem cells, which are of benefit from the viewpoint of reduced hospital stay, morbidity, and immune reactions, is a tool in a new clinical platform for a whole spectrum of intractable diseases. The TERM Received August 23, 2009; final acceptance October 16, 2010. Online prepub date: November 5, 2010. Address correspondence to Yoichi Yamada, Center for Genetic and Regenerative Medicine, Nagoya University School of Medicine, 65 Tsurumacho, Showa-ku, Nagoya 466-8550, Japan. Tel: 81-52-744-2348; Fax: 81-52-744-2352; E-mail: yyamada@med.nagoya-u.ac.jp 1003
1004 YAMADA ET AL. clinical trials for effective treatment of osseous defects, rich plasma (PRP), 3) canine mesenchymal stem cells/ and favorable results have been obtained (22 24), but PRP (cmscs/prp), 4) canine dental pulp stem cells/prp bone marrow aspiration is an invasive and painful proce- (cdpscs/prp), and 5) puppy deciduous tooth stem dure for the donor. Besides, the number, proliferation, cells/prp (pdtscs/prp), and investigated for os- and differentiation potential of MSCs decline with in- teogenesis. We created defects and implanted four materials creasing age (10). at random sites (Fig. 1). Previous studies have reported that several populations of stem cells have been isolated from dental tissues Isolation and Cultivation of MSCs, DPSCs, and DTSCs including the pulp of both exfoliated and adult teeth, The cmscs were isolated from the parent dog s iliac periodontal ligament, and dental follicle. Dental pulp crest marrow aspirate (10 ml), dental pulp tissues were stem cells (DPSCs) and stem cells from human exfoli- obtained from extracted puppy deciduous teeth and parent ated deciduous teeth (SHED) have generic mesenchymal permanent teeth (Fig. 1), and cmscs, cdpscs, and stem cell-like properties such as self-renewal and multi- pdtscs were isolated and cultured as previously described lineage differentiation (5,13,21). DPSCs and SHED (5,9,13,21). Briefly, the cmscs were incubated were demonstrated to have the ability to generate not at 37 C in a humidified atmosphere containing 95% air only dental tissue such as dentine/pulp-like complexes and 5% CO 2 and replated at densities of 3.1 10 3 cells/ but also bone tissue. Since SHED exhibit higher prolif- cm 2 in control medium; Dulbecco s modified Eagle s eration rates (1,5,15) and can be obtained with ease medium (DMEM; Gibco, Rockville, MD) containing compared to BMMSCs, they might become an attractive 20% mesenchymal cell growth supplement (Lonza source of autologous stem cells for bone regeneration. Inc., Walkersville, MD) and antibiotics (100 U/ml peni- MSCs have been reported to have a potent immunosup- cillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin pressive effect that inhibits the T cells, B cells, and NK B; Gibco). The pulp was gently removed and dipressive cells and dendritic cell function and be useful for the treat- gested in a solution of 3 mg/ml collagenase type I and ment of mesenchymal disorders. In a previous study, they 4 mg/ml dispase for 1 h at 37 C. After filtration using demonstrated that allogeneic BMMSCs offer feasible post- 70-mm cell strainers (Falcon, BD Labware, Franklin transplantation therapy for osteogenesis imperfect without Lakes, NJ), cells were cultured in the same medium at clinical symptoms, indicating the autoimmune response 37 C containing 95% air and 5% CO 2. After primary against the marrow microenvironment (6). On the other culture, the cells were subcultured at about 1 10 4 cells/ hand, DPSCs have been reported to have higher preven- cm 2. Three supplements for inducing osteogenesis, dexamethasone tion ability of T-cell alloreactivity and immunosuppressive (Dex), sodium β-glycerophosphate (β-gp), activity compared to BMMSCs (16). Because of their low and L-ascorbic acid 2-phosphate (AsAP) were purchased immunogenic potential, MSCs from dental pulp could be from Sigma Chemical Co. (St. Louis, MO). The cells used as a potential allogenic therapeutic tool for regenera- were incubated at 37 C in a humidified atmosphere containing tive medicine, particularly for bone regeneration. 95% air and 5% CO 2. In culture, cmscs, cdpscs, In this study, we utilized the canine mandible as a and pdtscs were trypsinized and used for implanting. large-animal model, in which mandibular defects were established for tissue engineering and therapies (14,24, PRP, PRP Gel Preparation, and Injection of cmscs/ 25), to examine the potential of using stem cells, autologous PRP, cdpscs/prp, pdtscs/prp Admixture MSCs, DPSCs, and allologous deciduous tooth PRP and the relevant condition were prepared as pre- stem cells (DTSCs). viously described (24). Briefly, approximately 50 ml whole blood was drawn from the canine into centrifuge MATERIALS AND METHODS tubes containing 10 ml culture medium with 250 U/ml Canine Animal Models preservative-free heparin. The blood was first centrifuged All protocols undertaken in this study were performed in a standard laboratory centrifuge, Himac CT in accordance with protocols approved by the (Hitachi Koki; Hitachi city, Japan). Subsequently, yellow Institutional Animal Care Committee and the university plasma (containing buffy coat, which contained committee (permission number 19373). After a period platelets and leukocytes) was taken up into a neutral monovette of housing, hybrid dogs were operated on under general with a long cannula. Second, centrifugation was anesthesia. The first molar, premolars, and the second performed to combine the platelets into a single pellet and third premolars in the mandible region were extracted. and the plasma supernatant, which is platelet-poor Bone defects on both sides of the mandible were plasma (PPP) and contains relatively few cells, was re- prepared with a trephine bar with a diameter and depth moved. The resulting pellet of platelets, buffy coat/ of 10 mm (25). The defects were implanted with graft plasma fraction (PRP), was resuspended in the residual materials as follows: 1) control (defect only), 2) platelet- 5 ml plasma and used in platelet gel. PRP was stored
BONE REGENERATION USING PROMISING STEM CELLS 1005 Figure 1. Schema of experimental protocol. Magnification of cell morphology (cmscs, cdpscs, and pdtscs) ( 40). for about 1 day at room temperature in a conventional rated into a 5-ml syringe, and in a second 2.5-ml syringe shaker until use. Bovine thrombin in powder form 500 µl thrombin/calcium chloride mixture was aspirated, (5,000 units) was dissolved in 5 ml of 10% calcium and the cells were resuspended directly in PRP. The two chloride in a separate sterile cup. Next, 3.5 ml PRP, syringes were connected with a T connector and the these cells (1.0 10 7 cells/ml), and 0.5 ml air were aspi- plungers of the syringes were pushed and pulled alterna-
1006 YAMADA ET AL. tively, allowing air bubble to transverse the two syrin- sal kit (Ventana) according to the manufacturer s instructions ges. The contents assumed a gel-like consistency as the for immunohistochemical staining. thrombin affected the polymerization of fibrin to produce an insoluble gel. The gel was injected into the bone Statistical Analysis defect field using a 5-ml syringe. Group means and SDs were calculated for each measured parameter. Differences in newly formed bone be- Histological and Histomorphometric Analysis tween the control, PRP, cmscs/prp, cdpscs/prp, and Each implantation site was excised with a trephine pdtscs/prp groups were analyzed using the Tukeybar of 2 mm diameter at 2, 4, and 8 weeks after implan- Kramer test following one-way analysis of variance tation, and sections for each experimental group were (ANOVA). A value of p < 0.05 or p < 0.001 indicated assessed by histological and histomorphometrical meth- statistical significance. ods as previously (25). The specimens were fixed in 10% buffered formalin, decalcified (K-CX; Falma Co., RESULTS Tokyo), and stained with hematoxylin and eosin. These specimens were examined under a light microscope and Macro Findings and Histological Evaluation of the analyzed by a pathologist, blinded to the identity of each Implants (PRP, cmscs/prp, cdpscs/prp, and specimen, who determined the presence or absence of pdtscs /PRP) Compared to the Control In Vivo bone formation. The experimental design of 10-mm-long defects created Histomorphometrical findings were analyzed with a in an established canine mandible model (15,25,26) microcomputer for image analysis. Each image of the was implanted with PRP, cmscs/prp, cdpscs/prp, specimens at the implantation site excised with a trephine and pdtscs /PRP (Figs. 1 and 2). Macroscopic findings bar of 2 mm diameter was copied on color rever- showed that bone regeneration using cmscs/prp, sal film, digitized as a 256 256 array of 8-bit density cdpscs/prp, and pdtscs/prp was to a natural mar- values, and transferred to a microcomputer for analysis ginal bone level, but regeneration using PRP and the (NIH Image, version 1.61; National Institutes of Health) control (defect only) was not complete (Fig. 2). In the (20). The augmented area was defined as the area en- pdtscs/prp group, no symptoms of pdtscs rejection closed within the mandible bone excised with a trephine were observed in the parent recipient mandible (Fig. 2). bar of 10-mm diameter. The volume of newly formed Bone regeneration and implant resorption were also bone in the augmented area was quantified using this monitored by histological evaluation at 2, 4, and 8 computer-based image analysis system. This was calcu- weeks. Osteogenesis spread slowly through the defect lated as the representative area percent of bone present base in PRP and controls. In contrast, defects filled with by deducting normal bone areas from measurement ar- cmscs/prp, cdpscs/prp, and pdtscs/prp implants eas in the section based on 2-mm biopsies. were found to show good bone formation, suggesting that bone formation occurred at about the same rate as Immunofluorescence and Immunohistochemical Analysis implant resorption. Implanted and nonimplanted control Eight weeks after implantation, specimens were fixed regions were collected at 2, 4, and 8 weeks, and proin 10% buffered formalin, decalcified (K-CX; Falma cessed and decalcified for histology. Co.), and used for immunofluorescence and immunohistochemical In PRP and controls, there were few bone formation analysis. The sections were incubated for 32 and the defects were filld by fibrous tissue evan at 2, 4, min at 40 C with primary mouse anti-bovine osteocalcin and 8 weeks (Figs. 3A F, 4A F). On the other hand, monoclonal antibody (Takara Bio. Inc., Shiga, Japan) defects filled with cmsc/prp, cdpscs/prp, and pdtscs/ diluted 1:200 in PBS. Sections were then washed three PRP resulted in new bone formation with active osteo- times in PBS, and incubated overnight at 4 C with goat cytes even after 4 weeks, and matured bone formation anti-mouse IgG fluorescein-conjugated secondary antibody with a well lamellar bone and abundant marrow cavity (Chemicon International, Inc., Billerica, MA, USA) were seen at 8 weeks (Figs. 3G O, 4G O), but the for- diluted 1:200 in PBS for immunofluorescence staining. mation by pdtscs/prp was slightly weak compared Finally, the sections were washed in PBS and mounted with cmscs/prp and cdpscs/prp (Figs. 3, 4). with Vectashield with DAPI (Vector Laboratories Inc., Immunohistological analysis revealed that mineral- Burlingame, CA, USA). On the other hand, the sections ized tissues and osteoblasts in cmscs/prp, cdpscs/ were incubated for 8 min at 40 C with rabbit anti-mouse PRP, and pdtscs/prp specimens were positive for os- IgG biotin-conjugated secondary antibody using a Ven- teocalcin 8 weeks after implantation (Fig. 5). On the tana NexES IHC (Ventana, Tucson, AZ, USA) automated other hand, positive staining was not detected in control slide staining system and an iview DAB Univer- and PRP specimens (Fig. 5B, C, E, F).
BONE REGENERATION USING PROMISING STEM CELLS 1007 Figure 2. Macroscopic observations for bone regeneration. (A) The canine mandible healed after extraction. (B) Experimental design in the mandible prepared with a trephine bar of 10-mm diameter. (C) Representative implanted materials in bone defects. L1, defect only; L2, cmscs/prp; L3, pdtscs/prp. (D) New bone regeneration with cmscs/prp, pdtscs/prp, and control groups at 2 weeks. L1, defect only; L2, cmscs/prp; L3, pdtscs/prp. (E) New bone regeneration with cmscs/prp, pdtscs/prp, and control groups at 4 weeks. L1, defect only; L2, cmscs/prp; L3, pdtscs/prp. (F) New bone regeneration with cmscs/prp, pdtscs/prp, and control groups at 8 weeks. L1, defect only; L2, cmscs/prp; L3, pdtscs/prp. Using cmscs/prp and pdtscs/ PRP, bone was regenerated to a natural level, but regeneration in the control (defect only) was not complete. Histomorphometric Analysis data; however, there was no significant difference in newly formed bone between cmscs/prp, cdpscs/prp, The bone-regenerating ability of all implants was assessed and pdtscs /PRP over time (Table 1). by measuring cortical and medullary bone-regen- erated areas by image analysis (Table 1). Adding PRP DISCUSSION to the cavity did not significantly increase the cortical TERM by stem cells with minimal invasiveness have or medullary bone-regenerated area compared to the been attempted for clinical applications and could lead control. In contrast, cmscs/prp and cdpscs/prp to good clinical results (22 24). Various stem cells, ES groups showed a significant increase in the regenerated cells, ips cells, or somatic stem cells have been identi- area compared with the control (p < 0.05 at 4 weeks, fied, but the main stem cells in clinical application p < 0.001 at 8 weeks, ANOVA) or cdpscs/prp (p < would be somatic stem cells. In this study, we therefore 0.001 at 8 weeks, ANOVA), confirming the histological focused on stem cells, DTSCs and DPSCs, which are
1008 YAMADA ET AL. Figure 3. Histologic evaluation of control, PRP, cmscs/prp, cdpscs/prp, and pdtscs /PRP implantations each week (lower magnification). Sections of representative implants are shown for the respective group. The sections were stained by hematoxylin and eosin. Original magnification 40 for all prints. (A) Two weeks in control group, (B) 4 weeks in control group, (C) 8 weeks in control group, (D) 2 weeks in PRP group, (E) 4 weeks in PRP group, (F) 8 weeks in PRP group, (G) 2 weeks in cmscs/prp group, (H) 4 weeks in cmscs/prp group, (I) 8 weeks in cmscs/prp group, (J) 2 weeks in cdpscs/prp group, (K) 4 weeks in cdpscs/prp group, (L) 8 weeks in cdpscs/prp group. (M) 2 weeks in pdtscs/prp group, (N) 4 weeks in pdtscs/prp group, (O) 8 weeks in pdtscs/prp group.
BONE REGENERATION USING PROMISING STEM CELLS 1009 Figure 4. Histologic evaluation of control, PRP, cmscs/prp, cdpscs/prp, and pdtscs/prp implantations each week (higher magnification). Sections of representative implants are shown for the respective group. The section was stained by hematoxylin and eosin. Original magnification 200 for all prints. (A) Two weeks in control group, (B) 4 weeks in control group, (C) 8 weeks in control group, (D) 2 weeks in PRP group, (E) 4 weeks in PRP group, (F) 8 weeks in PRP group, (G) 2 weeks in cmscs/prp group, (H) 4 weeks in cmscs/prp group, (I) 8 weeks in cmscs/prp group, (J) 2 weeks in cdpscs/prp group, (K) 4 weeks in cdpscs/prp group, (L) 8 weeks in cdpscs/prp group. (M) 2 weeks in pdtscs/prp group, (N) 4 weeks in pdtscs/prp group, (O) 8 weeks in pdtscs/prp group.
1010 YAMADA ET AL. Figure 5. Immunofluorescence and immunohistochemical analysis of osteocalcin at 8 weeks after implantation. (A, D, G, J, M) Hematoxylin and eosin staining. (B, E, H, K, N) Immunohistochemical staining of osteocalcin. (C, F, I, L, O) Immunofluorescence staining of osteocalcin. Higher magnification of the rectangular areas in (A, D, G, J, and M) is shown in (B, C, E, F, H, I, K, L, N, and O). Scale bars: 100 µm (A, D, G, J, M); 50 µm (B, C, E, F, H, I, K, L, N, O).
BONE REGENERATION USING PROMISING STEM CELLS 1011 Table 1. Histomorphology Data 2 Weeks (%) 4 Weeks (%) 8 Weeks (%) * * * * 19.0 ± 2.9 Control 6.9 ± 5.3 17.7 ± 2.6 PRP 7.8 ± 2.8 19.8 ± 2.8 19.7 ± 6.0 cmscs/prp 15.3 ± 3.2 35.6 ± 4.7 52.8 ± 3.5 cdpscs/prp 8.5 ± 1.8 37.4 ± 7.4 61.6 ± 1.3 pdtscs/prp 13.8 ± 6.3 25.5 ± 1.1 54.7 ± 2.2 *p < 0.05. p < 0.001. the most promising, and MSCs, which have been con- that SHED was able to produce woven bone and a dentin-like sidered the standard for stem cell sources in TERM, and structure (4,11,18). DPSCs have been considered investigated their bone regeneration ability compared to an appropriate candidate for dental tissue regeneration, scaffold (PRP) and defect only. The results indicated a dentin/pulp-like structure, and for the treatment of that newly formed bone areas containing these stem cells general diseases (2,8); however, little is known about were 19.0 ± 2.9% (control), 19.7 ± 6.0% (PRP), 52.8 ± the differences between SHED and DPSCs. In this 3.5% (cmscs/prp), 61.6 ± 1.3% (cdpscs/prp), and study, we found that DPSCs/PRP and DTSCs/PRP, in 54.7 ± 2.2% (pdtscs/prp) at 8 weeks histometrically, spite of an allograft, showed bone regeneration capacity and cmscs/prp, cdpscs/prp groups (at 4 and 8 similar to that of MSCs/PRP (Figs. 2 5). This might be weeks), and pdtscs/prp (at 8 weeks) showed a significant based on a similar stem cell origin and possessing the increase in area compared with the control and common properties of MSCs. PRP, confirming the histological data (Figs. 3 5, Table Moreover, it was stated that MSCs could be used to 1). However, there was no significant difference in prevent immune complications related to both hematopoietic newly formed bone among cmscs/prp, cdpscs/prp, stem cells and solid organ transplantation, and it and pdtscs /PRP (Table 1). And PRP contains various was proposed that MSCs are suppressors of immune activity growth factors, such as platelet-derived growth factors (16,19), because MSCs do not induce T-cell allogrowth (PDGF), transforming growth factor-β (TGF-β), and in- reactivity and display an immunoregulatory capacity by sulin-like growth factor-i (IGF-I) (12), but PRP alone suppressing T-cell responses in vitro and in vivo (3, did not allow osteogenesis to occur in the affected areas 16,27). On the other hand, the immunosuppressive activity (Figs. 3 5, Table 1). In short, the PRP scaffold for of human DPSCs was significantly higher than by cmscs, cdpscs, and pdtscs would encourage these bone marrow (16). Accordingly, we did not use any protocol cells adhesion, proliferation, and differentiation to elicit for immunosuppression in our experimental anicells bone formation. The disappearance of the cmscs/prp, mals as for allograft using DTSCs. The results showed cdpscs/prp, and pdtscs/prp left in place induced that no clinical symptoms of pdtsc rejection were ob- bone tissue formation, which then self-organized accord- served in the parent recipient canines and the parents did ing to the surrounding environment. not reject pediatric DTSCs in this study. It is important Immunohistochemical analysis of osteocalcin, which to address whether these DTSCs trigger the immunologic is a specific marker of osteogenesis, indicated that regenerative system of the recipients; therefore, successful bone regen- tissues in cmscs/prp, cdpscs/prp, and eration by allogenic DTSCs might be related to immuno- pdtscs/prp specimens were positive for osteocalcin suppressive activity by stem cells from dental pulp. and therefore identified as bone (Fig. 5). In a previous In conclusion, DPSCs and DTSCs from dental pulp study, DSPCs were considered to possess multipotent could get a good bone formation the same as MSCs ability and stem cell-like properties, such as self-renewal from bone marrow through natural bone formation process. capability and multilineage differentiation, and the phenotypes Furthermore, out results indicate that DTSCs do were similar to those of bone marrow-derived not cause allogeneic graft rejection. Hence, promising MSCs (5,21); therefore, bone regeneration by DPSCs DPSCs and DTSCs would apply as a broad potential with TERM might resemble that by MSCs. stem cell source for bone regeneration in various bone On the other hand, stem cells from human exfoliated surgeries, such as oral maxillofacial surgery, plastic surdeciduous teeth (SHED), DTSCs, was identified as a gery, craniofacial anomalies, and orthopedics. novel population of stem cells capable of differentiating into various cell types, such as osteoblasts, odontoblasts, ACKNOWLEDGMENTS: The authors wish to thank Wataru adipocytes, and neural cells (4,5,13). It was suggested Katagiri, Ryoko Yoshimi, Kazuto Okabe, Tomoyuki Kohgo,
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