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Allogeneic Bone Marrow Mesenchymal Stem Cell Transplantation for Periodontal Regeneration J. Du, Z. Shan, P. Ma, S. Wang and Z. Fan J DENT RES 2014 93: 183 originally published online 13 November 2013 DOI: 10.1177/0022034513513026 The online version of this article can be found at: http://jdr.sagepub.com/content/93/2/183
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93210.1177/0022034513513026
RESEARCH REPORTS Biomaterials & Bioengineering
J. Du1,2, Z. Shan1,2, P. Ma2, S. Wang2,3, and Z. Fan1* 1
Laboratory of Molecular Signaling and Stem Cells Therapy, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, Beijing 100050, China; 2Molecular Laboratory for Gene Therapy and Tooth Regeneration, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, Beijing 100050, China; and 3Department of Biochemistry and Molecular Biology, Capital Medical University School of Basic Medical Sciences, Beijing 100069, China; *corresponding author, [email protected]
Allogeneic Bone Marrow Mesenchymal Stem Cell Transplantation for Periodontal Regeneration
J Dent Res 93(2):183-188, 2014
Abstract
In this study, we investigated the possibility of using local administration of allogeneic bone marrow mesenchymal stem cells (BMMSCs) to induce tissue regeneration in periodontal defects in a rat model of periodontitis. BMMSCs isolated from rats were mixed with 0.9% NaCl solution and injected into periodontal defects. Control groups were 0.9% NaCl solution or left untreated. The clinical assessments, x-rays, and histological examinations were used to evaluate the effect. At 12 wks post-transplantation, quantitative analysis revealed average probing bone loss values of 1.2 ± 0.19, 1.6 ± 0.2, and 1.7 ± 0.14; the bone regeneration rate was 53%, 45%, and 44% in the BMMSC+NaCl group, NaCl group, and untreated group, respectively. The clinical assessments, x-rays, and histological examinations revealed significant periodontal tissue regeneration in the BMMSC injection group, compared with the control groups. The ELISA results showed that TNFα, IFNγ, and IL1β were 2,674.88 ± 102.77 pg/mL vs. 3,422.1 ± 51.98 pg/mL, 609.85 ± 25.5 pg/mL vs. 803.79 ± 33.85 pg/mL, and 1,038.46 ± 76.29 pg/mL vs. 1,175.26 ± 105.55 pg/mL in the BMMSC+NaCl group and NaCl group, respectively, indicating that BMMSC injection inhibited the inflammatory factors TNFα, IFNγ, and IL1β. Our results indicate that local administration of BMMSCs can repair defects due to periodontitis, exerting anti-inflammatory and immunomodulatory functions.
KEY WORDS: stem cells, periodontitis, injection, inflammation, immune, rats. DOI: 10.1177/0022034513513026 Received June 27, 2013; Last revision October 20, 2013; Accepted October 21, 2013 A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental. © International & American Associations for Dental Research
Introduction
P
eriodontitis is one of the most widespread inflammatory diseases in humans, involving destruction of the supporting structures of the teeth, including the periodontal ligament, bone, and gingival tissues. It is the main pathogenic cause of adult tooth loss, especially among the elderly (Kinane and Bartold, 2007). Periodontitis is also associated with a number of systemic diseases, such as diabetes and cardiovascular disease (Petite et al., 2000; Bianco et al., 2001). Restoration of lost teeth and regeneration of periodontal tissue are key focuses of dental clinicians and researchers. To date, the treatment of bone defects resulting from periodontitis remains challenging. Animal model studies and clinical trials have tested several approaches to periodontal disease treatment and periodontal tissue regeneration, including applications of enamel matrix derivative (EMD) (Kémoun et al., 2007) and various growth factors (GF), such as basic fibroblast growth factor (bFGF), transforming growth factor-β (TGF-β), etc. (Oi et al., 2009; Maeda et al., 2013). However, some problems remain unresolved, for example, the short half-time life of growth factors. Moreover, in local tissue, growth factors are diluted and metabolized very quickly. Therefore, high doses of growth factors must be applied to obtain ideal effects. Mesenchymal stem cell (MSC)-mediated tissue engineering is a promising technique for tissue regeneration (Petite et al., 2000). Our research group previously generated a swine model of periodontitis. In this model, we cured periodontitis and induced significant periodontal tissue regeneration using periodontal ligament stem cells (PDLSCs) mixed with HA/TCP scaffolds (Liu et al., 2008). This method requires surgery, which can be traumatic for patients; thus, such treatment is suitable only for patients with severe periodontitis and without severe systemic diseases. Moreover, the clinical application of this approach is largely impeded by the limited availability of PDLSCs, particularly for elderly patients. Bone marrow mesenchymal stem cells (BMMSCs) are easily accessible and capable of generating robust amounts of bone in vivo and have thus emerged as an important cell source for bone regeneration (Mankani et al., 2006). Such use of BMMSCs has been clinically demonstrated in femur fracture repair and regeneration (Shao et al., 2006). Recent studies have also shown that BMMSCs can ameliorate diabetes in animal models (Fiorina et al., 2009) and accelerate wound closure (Wu et al., 2007). BMMSCs are pluripotent stem cells capable of differentiating into several cell types.
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J Dent Res 93(2) 2014 influence host cells to restore tissue. Therefore, it is important to investigate the potential immune responses caused by exogenous stem cells. In the present study, we investigated the possibility of using local injection of allogeneic BMMSCs for regenerating periodontal tissues that are damaged by periodontitis, and their effect on inflammation and immune response. Our results demonstrated that local injection of BMMSCs repaired tissue defects due to periodontitis and had anti-inflammatory effects in the rat model, thus showing that this method is potentially an easy and non-invasive therapy for clinical periodontitis.
Materials & Methods Animals This study was reviewed and approved by the Animal Care and Use Committee of Capital Medical University, Beijing, China. Twenty-seven female SD rats (4-5 wks old; 80-100 g) were obtained from the Institute of Animal Science of the Vital River Co., Ltd., Beijing, China. They were fully barrier-reared with free access to water and a regular supply of food.
Figure 1. Application of BMMSCs for periodontitis treatment. (A) Each periodontal defect in SD rats was injected at 3 gingival sites: (a) the mesial side of the molar, (b) the distal side of the molar, and (c) the middle of the molar. (B) Probing bone loss was evaluated by measurement of the distance between the cement/enamel junction and alveolar bone crest at 5 sites in each defect: (a) the middle of the mesial root, (b) the middle of the distal root, (c) the middle of the middle root, (d) the middle of the mesial furcation, and (e) the middle of the distal furcation. (C) The apex–cusp (AC) and apex–alveolar ridge (AR) distances were measured.
Kuo et al. (2011) reported that BMMSCs can promote the formation of periodontal ligament, odontoblast, and cementum from dental pulp stem cells (DPSCs). Another research group also showed that bone marrow progenitor cells can communicate with dental tissues (Zhou et al., 2011). Kawaguchi et al. (2004) used auto-transplanted BMMSCs with atelocollagen to regenerate periodontal tissue, cementum, and alveolar bone. It has also been shown that BMMSCs can stimulate proliferation of host progenitor cells or various chemotaxic molecules to stimulate local tissue (Wang et al., 2008). These findings suggest that injected BMMSCs might be able to differentiate into multi-lineage tissues in vivo. Jeong et al. (2012) recently found that autoimmune diseases can be aggravated by antigens from Porphyromonas gingivalis. But the molecular mechanism by which innate and adaptive immune systems contribute to inflammation in autoimmune diseases is not yet understood. BMMSCs reportedly possess an immunomodulatory function that promotes successful treatment of immune diseases (Yamaza et al., 2010). These associations led us to wonder whether immunomodulatory activity was also present following BMMSC injection. Stem cell research is one of the most promising areas of research in dentistry. However, there are still many issues to be addressed. Thus far, stem cells have been shown to have the ability to modulate the responses of the immune system and
Establishment of a Rat Model of Periodontitis and Local Injection of BMMSCs In 17 rats, we generated periodontal defects caused by periodontitis by binding wire around the bilateral maxillary first molars and subsequently inoculating them with Porphyromonas gingivalis (strain 381) (Chang et al., 1994). After 4 wks, 2 rats were randomly selected and examined radiographically and by histological examinations to guarantee periodontal defect formation. In total, 30 periodontal defects were obtained in the remaining 15 rats. An additional 5 rats were used as healthy controls. These defects were randomly assigned to 3 groups: one group with no treatment; one group with an injection of 0.9% NaCl at 3 sites (the mesial side of the molar, the distal side of the molar, and the middle of the molar); and one group with an injection of allogeneic BMMSCs+0.9% NaCl at 3 sites (the mesial side of the molar, the distal side of the molar, and the middle of the molar, with each group including 10 defects in 5 rats). For BMMSC treatment, each site of the defect was injected with 1×106 cells BMMSCs in 0.1 mL 0.9% NaCl (Fig. 1A). The needle was inserted from the mucosa to the bone surface (supraperiosteally), and the cell mixture was injected after significant resistance was encountered.
Clinical Assessment of Probing Bone Loss For assessment of probing bone loss (PBL), rats were sacrificed with CO2, the maxillary bone was dissected, and the soft tissue was removed. With the use of a stereoscopic microscope (SZX12, Olympus, Tokyo, Japan), the specimen was placed perpendicularly to the occlusal plane of the maxilla. Forty photographs were taken at 16× magnification and analyzed with Adobe Photoshop CS6 (Adobe, San Jose, CA, USA). The PBL was evaluated by measurement of the distance between the cement-enamel junction and the alveolar bone crest at 5 sites for each defect: the middle of the mesial root, the middle of the
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J Dent Res 93(2) 2014 185 Allogeneic BMMSC Transplantation for Periodontal Regeneration
Figure 2. Clinical and x-ray assessment of bone tissue regeneration. (A) Intra-oral photographs at 12 wks after transplantation, showing better bone tissue regeneration in the BMMSC+NaCl administration group than in the NaCl or untreated group. (B) Soft x-ray showed only limited bone formation in the untreated and NaCl groups, while alveolar bone regeneration was observed in the BMMSCs+NaCl group. (C-H) At 5 molar sites in 4 groups, probing bone loss (PBL) was lowest in the BMMSC+NaCl group compared with that in untreated and NaCl groups. (I) Bone regeneration rate (BRR) was calculated as [apex–cusp distance/apex–alveolar ridge distance]×100%. BRR was highest in the BMMSC+NaCl group compared with that in untreated and NaCl groups, indicating more bone tissue regeneration with BMMSC+NaCl injection than in the NaCl or untreated group. Statistical significance was evaluated by analysis of variance. All error bars represent SD (n = 10). *p < .05. **p < .01.
distal root, the middle of the middle root, the middle of the mesial furcation, and the middle of the distal furcation. All measurements at each site were made along the long axis of the root in the photograph, with scale tools provided by Photoshop (Fig. 1B). The PBL was calculated at the 5 sites for each molar, with a higher value representing more periodontal bone loss.
Measurement of Bone Regeneration Rate At 12 wks after injection, the bone regeneration rates (BRR) of the maxillary molars were examined by soft x-ray to measure the intrabony defects in 4 groups: healthy control, one group with no treatment, one group with injection of 0.9% NaCl, and
one group with injection of allogeneic BMMSCs+0.9% NaCl. The maxillary bone with the first molar was attached to a plastic board on top of an x-ray film (Kodak, Rochester, NY, USA). The specimen was placed perpendicular to the board to facilitate measurement of the height of the molar from the occlusal surface to the apical root and the alveolar ridge. The radiograph was exposed for 20 sec in an x-ray machine (Kyodo DMK 14z) at 30 kV and 2.5 mA and developed in an x-ray processor for 3 min. All measurements of the distal aspects of the first molar were performed with Photoshop software. The apex–cusp (AC) and apex–alveolar ridge (AR) distances were measured, and BRR was calculated as AR/AC×100% (Fig. 1C). Higher BRR values indicated less bone formation.
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Figure 3. HE staining showed new periodontal tissue regeneration in the periodontal defects of the BMMSC+NaCl group, while bone and periodontal ligament fiber shortages remained in the untreated and NaCl groups. The alveolar bone height in the BMMSC+NaCl group was also much larger than those in the untreated and NaCl groups. D, dentin; B, bone; PDL, periodontal ligament; CEJ, cemento-enamel junction; HAB, height of alveolar bone. Scale bar = 100 µm.
Statistical Analysis All statistical calculations were performed with SPSS13 statistical software. Statistical significance was determined by the independent sample test or analysis of variance. A p value ≤ .05 was considered significant.
Results Local Injection of Allogeneic BMMSCs Improved Tissue and Bone Regeneration in Periodontitis At 12 wks post-injection, intra-oral photographs showed that the BMMSC+NaCl injection group had better periodontal tissue regeneration than the NaCl and untreated groups, but less regeneration than the healthy controls (Fig. 2A). Quantitative analysis revealed the following PBL values at the mesial site: 0.84 ± 0.06 in the healthy group, 1.52 ± 0.06 in the BMMSC+NaCl injection group, 2.07 ± 0.18 in the NaCl injection group, and 2.18 ± 0.19 in the untreated group. The PBL values at the distal site were: 0.75 ± 0.05 in healthy group, 1.2 ± 0.15 in the BMMSC+NaCl injection group, 1.67 ± 0.09 in the NaCl injection group, and 1.68 ± 0.13 in the untreated group. PBL values at the mesial furcation site were: 0.79 ± 0.03 in the healthy group, 1.38 ± 0.04 in the BMMSC+NaCl injection group, 1.52 ± 0.12 in the NaCl injection group, and 1.63 ± 0.16 in the untreated group. At the distal furcation site, the PBL values were: 0.8 ± 0.04 in the healthy group, 1.06 ± 0.07 in the BMMSC+NaCl injection group, 1.39 ± 0.1 in the NaCl injection group, and 1.38 ± 0.09 in the untreated group. PBL values at the middle site measured: 0.84 ± 0.03 in the healthy group, 1.31 ± 0.13 in the BMMSC+NaCl injection group, 1.56 ± 0.14 in the NaCl injection group, and 1.68 ± 0.13 in the untreated group (Figs. 2C-2G). The average PBL values were 0.8 ± 0.21 in the healthy group, 1.2 ± 0.19 in the BMMSC+NaCl injection group, 1.6 ± 0.2 in the NaCl injection group, and 1.7 ± 0.14 in the untreated group (Fig. 2H). Statistical analysis indicated that, although local BMMSC injection could not restore tissues to healthy levels, it significantly
J Dent Res 93(2) 2014 improved periodontal tissue regeneration compared with NaCl injection or no treatment. Soft x-ray results showed more bone formation in the BMMSC+NaCl injection group compared with that in the NaCl and untreated groups, but formation did not reach normal levels (Fig. 2B). Quantitative analysis revealed that the BRR was 53% in the BMMSC+NaCl injection group, 45% in the NaCl injection group, and 44% in the untreated group (Fig. 2I), indicating that local BMMSC injection enhanced bone formation in periodontal defects. Histopathological results showed that the new bone and periodontal ligament in the periodontal defects in the BMMSC+NaCl group were regenerated to the highest levels compared with those in the NaCl and untreated groups. In contrast, the NaCl and untreated groups still showed typical periodontitis status, including deep periodontal pockets and shortage of new bone and periodontal ligament fibers (Fig. 3).
Local Injection of BMMSCs Inhibited the Expressions of TNFα, IFNγ, and IL1β The ELISA results showed that the concentration of TNFα was 2,674.88 ± 102.77 pg/mL in the BMMSC+NaCl group, 3,422.1 ± 51.98 pg/mL in the NaCl group, and 870.99 ± 28.02 pg/mL in the healthy group. IFNγ was 609.85 ± 25.5 pg/mL in the BMMSC+NaCl group, 803.79 ± 33.85 pg/mL in the NaCl group, and 212.88 ± 63.68 pg/mL in the healthy group; and IL1β was 1,038.46 ± 76.29 pg/mL in the BMMSC+NaCl group, 1,175.26 ± 105.55 pg/mL in the NaCl group, and 91.93 ± 38.14 pg/mL in the healthy group (Figs. 4A-4C). However, the concentration of IL6 was not significantly different (Fig.4D). The results showed that although TNFα, IFNγ, and IL1β could not be restored to healthy levels, their concentrations were significantly decreased in the periodontitis supernatants after BMMSC injection compared with those in the NaCl group, indicating an anti-inflammatory effect of local BMMSC injection in rat periodontitis.
Discussion Key features of periodontal disease include alveolar bone loss and gingival atrophy. For years, researchers have searched for methods to restore periodontal tissue and attachment. In previous studies, the delivery of stem cells to periodontal wounds involved both biomaterial-based and biomaterial-free approaches. Biomaterial systems included collagen sponges (Kawaguchi et al., 2004), gelatin (Kuo et al., 2008), or HA/TCP scaffolds (Liu et al., 2008) or similar matrices. Biomaterial-free methods involved cell sheet engineering (Wei et al., 2012) or cell pellets (Yang et al., 2009). The transplantation format depends on the surgical procedure. However, these treatments are suitable only for patients with severe periodontitis who are in a sufficient physical condition to tolerate surgery. Cell injection therapy is the most commonly used approach for treating a variety of diseases, including the regeneration of spinal cord injury (Koda et al., 2005) or myocardial infarction (Shake et al., 2002). Injected cells can be administered directly to defective tissues. The usefulness of BMMSCs can be attributed
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J Dent Res 93(2) 2014 187 Allogeneic BMMSC Transplantation for Periodontal Regeneration to their low immunogenicity and their immunomodulatory function. Thus, in the present study, we investigated the feasibility of BMMSC suspension injection in a rat periodontitis model. Initially, we observed lesion restoration in the untreated group, the NaCl injection group, and the BMMSC suspension group. According to clinical observation and radiographic and histological examination, we found that the BMMSC injection group contributed significantly more to periodontal tissue repair than did the untreated or the NaCl injection group. Although the main drawback of cell suspension injection is a high cell number loss and the unknown fate of BMMSCs in the local tissues, the results of this study demonstrated that the cells can work efficiently in the local tissue. In addition, animal experiments demonstrated that transplanted BMMSCs labeled with green fluorescent protein Figure 4. Local administration of BMMSCs inhibited TNFα, IFNγ, and IL1β expression. TNFα (GFP) were detectable by immunohisto(A), IFNγ (B), and IL1β (C) concentrations were significantly decreased in the periodontitis supernatants after BMMSCs+NaCl injection compared with those in the NaCl group. chemical analysis 4 wks after transplanStatistical significance was determined with the independent sample test. (D) No significant tation (Kawaguchi et al., 2004). differences of IL-6 were found among these groups. All error bars represent SD (n = 6). T-cell-mediated immunity is consid*p < .05. **p < .01. ered a critical factor in periodontitis pathogenesis. Gaffen and Hajishengallis System (2013-3-035 to Z.P.F.). The authors declare no potential (2008) described the Th (T-helper) 1/Th2 cytokine profile in conflicts of interest with respect to the authorship and/or publichronic periodontitis in humans. Our present results showed that cation of this article. the local injection of BMMSCs inhibited the expression of TNFα, IFNγ, and IL1β. These 3 cytokines play important roles in inflammation and immunity. IFNγ is produced by T-cells and enhances References T-cell-mediated immunity (Page et al., 1997). TNFα is reportedly Abramson SB, Yazici Y (2006). Biologics in development for rheumatoid involved in inflammatory response, bone loss, and autoimmune arthritis: relevance to osteoarthritis. Adv Drug Deliv Rev 58:212-225. diseases, including Crohn’s disease, ulcerative colitis, and multiAndreakos E (2003). Targeting cytokines in autoimmunity: new approaches, ple sclerosis (Andreakos, 2003; Abramson and Yazici, 2006). new promise. Expert Opin Biol Ther 3:435-447. Garlet et al. (2010) previously showed that inhibiting Treg funcBianco P, Riminucci M, Gronthos S, Robey PG (2001). Bone marrow strotion can increase IFNγ and TNFα levels and enhance alveolar mal stem cells: nature, biology, and potential applications. Stem Cells 19:180-192. bone loss and inflammatory cell migration. However, further Chang KM, Ramamurthy NS, McNamara TF, Evans RT, Klausen B, Murray investigations will be carried out to elucidate local immunological PA, et al. (1994). Tetracyclines inhibit Porphyromonas gingivalischanges in the periodontitis animal model. induced alveolar bone loss in rats by a non-antimicrobial mechanism. Taken together, our present results indicate that local injecJ Periodontal Res 29:242-249. tion of BMMSCs can repair tissue defects in cases of periodonFiorina P, Jurewicz M, Augello A, Vergani A, Dada S, La Rosa S, et al. (2009). Immunomodulatory function of bone marrow-derived mesentitis, as well as exert anti-inflammatory and immunomodulatory chymal stem cells in experimental autoimmune type 1 diabetes. functions in a rat model. This technique is promising as an easy J Immunol 183:993-1004. and non-invasive therapy for clinical periodontitis. Gaffen SL, Hajishengallis G (2008). A new inflammatory cytokine on the
Acknowledgments This work was supported by grants from the National Basic Research Program of China (No. 2010CB944801), the National Natural Science Foundation of China (81271101 to Z.P.F., 81070843 to Z.C.S.), the Program for New Century Excellent Talents in University (NCET-12-0611 to Z.P.F.), the Beijing Municipal Science and Technology Commission (Z1211000 05212004 to S.L.W.), and High-level Talents of Beijing Health
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Kémoun P, Laurencin-Dalicieux S, Rue J, Farges JC, Gennero I, ConteAuriol F, et al. (2007). Human dental follicle cells acquire cementoblast features under stimulation by BMP-2/-7 and enamel matrix derivatives (EMD) in vitro. Cell Tissue Res 329:283-294. Kinane DF, Bartold PM (2007). Clinical relevance of the host responses of periodontitis. Periodontol 2000 43:278-293. Koda M, Okada S, Nakayama T, Koshizuka S, Kamada T, Nishio Y, et al. (2005). Hematopoietic stem cell and marrow stromal cell for spinal cord injury in mice. Neuroreport 16:1763-1767. Kuo TF, Huang AT, Chang HH, Lin FH, Chen ST, Chen RS, et al. (2008). Regeneration of dentin-pulp complex with cementum and periodontal ligament formation using dental bud cells in gelatin-chondroitinhyaluronan tri-copolymer scaffold in swine. J Biomed Mater Res A 86:1062-1068. Kuo TF, Lin HC, Yang KC, Lin FH, Chen MH, Wu CC, et al. (2011). Bone marrow combined with dental bud cells promotes tooth regeneration in miniature pig model. Artif Organs 35:113-121. Liu Y, Zheng Y, Ding G, Fang D, Zhang CM, Bartold PM, et al. (2008). Periodontal ligament stem cell-mediated treatment for periodontitis in miniature swine. Stem Cells 26:1065-1073. Maeda H, Wada N, Tomokiyo A, Monnouchi S, Akamine A, et al. (2013). Prospective potency of TGF-β1 on maintenance and regeneration of periodontal tissue. Int Rev Cell Mol Biol 304:283-367. Mankani MH, Kuznetsov SA, Shannon B, Nalla RK, Ritchie RO, Qin Y, et al. (2006). Canine cranial reconstruction using autologous bone marrow stromal cells. Am J Pathol 168:542-550. Oi Y, Ota M, Yamamoto S, Shibukawa Y, Yamada S (2009). Beta-tricalcium phosphate and basic fibroblast growth factor combination enhances periodontal regeneration in intrabony defects in dogs. Dent Mater J 28:162-169. Page RC, Offenbacher S, Schroeder HE, Seymour GJ, Kornman KS (1997). Advances in the pathogenesis of periodontitis: summary of
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Allogeneic Bone Marrow Mesenchymal Stem Cell Transplantation for Periodontal Regeneration J. Du, Z. Shan, P. Ma, S. Wang and Z. Fan J DENT RES 2014 93: 183 originally published online 13 November 2013 DOI: 10.1177/0022034513513026 The online version of this article can be found at: http://jdr.sagepub.com/content/93/2/183
Published by: http://www.sagepublications.com
On behalf of: International and American Associations for Dental Research
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research-article2014
JDR
93210.1177/0022034513513026
RESEARCH REPORTS Biomaterials & Bioengineering
J. Du1,2, Z. Shan1,2, P. Ma2, S. Wang2,3, and Z. Fan1* 1
Laboratory of Molecular Signaling and Stem Cells Therapy, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, Beijing 100050, China; 2Molecular Laboratory for Gene Therapy and Tooth Regeneration, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, Beijing 100050, China; and 3Department of Biochemistry and Molecular Biology, Capital Medical University School of Basic Medical Sciences, Beijing 100069, China; *corresponding author, [email protected]
Allogeneic Bone Marrow Mesenchymal Stem Cell Transplantation for Periodontal Regeneration
J Dent Res 93(2):183-188, 2014
Abstract
In this study, we investigated the possibility of using local administration of allogeneic bone marrow mesenchymal stem cells (BMMSCs) to induce tissue regeneration in periodontal defects in a rat model of periodontitis. BMMSCs isolated from rats were mixed with 0.9% NaCl solution and injected into periodontal defects. Control groups were 0.9% NaCl solution or left untreated. The clinical assessments, x-rays, and histological examinations were used to evaluate the effect. At 12 wks post-transplantation, quantitative analysis revealed average probing bone loss values of 1.2 ± 0.19, 1.6 ± 0.2, and 1.7 ± 0.14; the bone regeneration rate was 53%, 45%, and 44% in the BMMSC+NaCl group, NaCl group, and untreated group, respectively. The clinical assessments, x-rays, and histological examinations revealed significant periodontal tissue regeneration in the BMMSC injection group, compared with the control groups. The ELISA results showed that TNFα, IFNγ, and IL1β were 2,674.88 ± 102.77 pg/mL vs. 3,422.1 ± 51.98 pg/mL, 609.85 ± 25.5 pg/mL vs. 803.79 ± 33.85 pg/mL, and 1,038.46 ± 76.29 pg/mL vs. 1,175.26 ± 105.55 pg/mL in the BMMSC+NaCl group and NaCl group, respectively, indicating that BMMSC injection inhibited the inflammatory factors TNFα, IFNγ, and IL1β. Our results indicate that local administration of BMMSCs can repair defects due to periodontitis, exerting anti-inflammatory and immunomodulatory functions.
KEY WORDS: stem cells, periodontitis, injection, inflammation, immune, rats. DOI: 10.1177/0022034513513026 Received June 27, 2013; Last revision October 20, 2013; Accepted October 21, 2013 A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental. © International & American Associations for Dental Research
Introduction
P
eriodontitis is one of the most widespread inflammatory diseases in humans, involving destruction of the supporting structures of the teeth, including the periodontal ligament, bone, and gingival tissues. It is the main pathogenic cause of adult tooth loss, especially among the elderly (Kinane and Bartold, 2007). Periodontitis is also associated with a number of systemic diseases, such as diabetes and cardiovascular disease (Petite et al., 2000; Bianco et al., 2001). Restoration of lost teeth and regeneration of periodontal tissue are key focuses of dental clinicians and researchers. To date, the treatment of bone defects resulting from periodontitis remains challenging. Animal model studies and clinical trials have tested several approaches to periodontal disease treatment and periodontal tissue regeneration, including applications of enamel matrix derivative (EMD) (Kémoun et al., 2007) and various growth factors (GF), such as basic fibroblast growth factor (bFGF), transforming growth factor-β (TGF-β), etc. (Oi et al., 2009; Maeda et al., 2013). However, some problems remain unresolved, for example, the short half-time life of growth factors. Moreover, in local tissue, growth factors are diluted and metabolized very quickly. Therefore, high doses of growth factors must be applied to obtain ideal effects. Mesenchymal stem cell (MSC)-mediated tissue engineering is a promising technique for tissue regeneration (Petite et al., 2000). Our research group previously generated a swine model of periodontitis. In this model, we cured periodontitis and induced significant periodontal tissue regeneration using periodontal ligament stem cells (PDLSCs) mixed with HA/TCP scaffolds (Liu et al., 2008). This method requires surgery, which can be traumatic for patients; thus, such treatment is suitable only for patients with severe periodontitis and without severe systemic diseases. Moreover, the clinical application of this approach is largely impeded by the limited availability of PDLSCs, particularly for elderly patients. Bone marrow mesenchymal stem cells (BMMSCs) are easily accessible and capable of generating robust amounts of bone in vivo and have thus emerged as an important cell source for bone regeneration (Mankani et al., 2006). Such use of BMMSCs has been clinically demonstrated in femur fracture repair and regeneration (Shao et al., 2006). Recent studies have also shown that BMMSCs can ameliorate diabetes in animal models (Fiorina et al., 2009) and accelerate wound closure (Wu et al., 2007). BMMSCs are pluripotent stem cells capable of differentiating into several cell types.
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J Dent Res 93(2) 2014 influence host cells to restore tissue. Therefore, it is important to investigate the potential immune responses caused by exogenous stem cells. In the present study, we investigated the possibility of using local injection of allogeneic BMMSCs for regenerating periodontal tissues that are damaged by periodontitis, and their effect on inflammation and immune response. Our results demonstrated that local injection of BMMSCs repaired tissue defects due to periodontitis and had anti-inflammatory effects in the rat model, thus showing that this method is potentially an easy and non-invasive therapy for clinical periodontitis.
Materials & Methods Animals This study was reviewed and approved by the Animal Care and Use Committee of Capital Medical University, Beijing, China. Twenty-seven female SD rats (4-5 wks old; 80-100 g) were obtained from the Institute of Animal Science of the Vital River Co., Ltd., Beijing, China. They were fully barrier-reared with free access to water and a regular supply of food.
Figure 1. Application of BMMSCs for periodontitis treatment. (A) Each periodontal defect in SD rats was injected at 3 gingival sites: (a) the mesial side of the molar, (b) the distal side of the molar, and (c) the middle of the molar. (B) Probing bone loss was evaluated by measurement of the distance between the cement/enamel junction and alveolar bone crest at 5 sites in each defect: (a) the middle of the mesial root, (b) the middle of the distal root, (c) the middle of the middle root, (d) the middle of the mesial furcation, and (e) the middle of the distal furcation. (C) The apex–cusp (AC) and apex–alveolar ridge (AR) distances were measured.
Kuo et al. (2011) reported that BMMSCs can promote the formation of periodontal ligament, odontoblast, and cementum from dental pulp stem cells (DPSCs). Another research group also showed that bone marrow progenitor cells can communicate with dental tissues (Zhou et al., 2011). Kawaguchi et al. (2004) used auto-transplanted BMMSCs with atelocollagen to regenerate periodontal tissue, cementum, and alveolar bone. It has also been shown that BMMSCs can stimulate proliferation of host progenitor cells or various chemotaxic molecules to stimulate local tissue (Wang et al., 2008). These findings suggest that injected BMMSCs might be able to differentiate into multi-lineage tissues in vivo. Jeong et al. (2012) recently found that autoimmune diseases can be aggravated by antigens from Porphyromonas gingivalis. But the molecular mechanism by which innate and adaptive immune systems contribute to inflammation in autoimmune diseases is not yet understood. BMMSCs reportedly possess an immunomodulatory function that promotes successful treatment of immune diseases (Yamaza et al., 2010). These associations led us to wonder whether immunomodulatory activity was also present following BMMSC injection. Stem cell research is one of the most promising areas of research in dentistry. However, there are still many issues to be addressed. Thus far, stem cells have been shown to have the ability to modulate the responses of the immune system and
Establishment of a Rat Model of Periodontitis and Local Injection of BMMSCs In 17 rats, we generated periodontal defects caused by periodontitis by binding wire around the bilateral maxillary first molars and subsequently inoculating them with Porphyromonas gingivalis (strain 381) (Chang et al., 1994). After 4 wks, 2 rats were randomly selected and examined radiographically and by histological examinations to guarantee periodontal defect formation. In total, 30 periodontal defects were obtained in the remaining 15 rats. An additional 5 rats were used as healthy controls. These defects were randomly assigned to 3 groups: one group with no treatment; one group with an injection of 0.9% NaCl at 3 sites (the mesial side of the molar, the distal side of the molar, and the middle of the molar); and one group with an injection of allogeneic BMMSCs+0.9% NaCl at 3 sites (the mesial side of the molar, the distal side of the molar, and the middle of the molar, with each group including 10 defects in 5 rats). For BMMSC treatment, each site of the defect was injected with 1×106 cells BMMSCs in 0.1 mL 0.9% NaCl (Fig. 1A). The needle was inserted from the mucosa to the bone surface (supraperiosteally), and the cell mixture was injected after significant resistance was encountered.
Clinical Assessment of Probing Bone Loss For assessment of probing bone loss (PBL), rats were sacrificed with CO2, the maxillary bone was dissected, and the soft tissue was removed. With the use of a stereoscopic microscope (SZX12, Olympus, Tokyo, Japan), the specimen was placed perpendicularly to the occlusal plane of the maxilla. Forty photographs were taken at 16× magnification and analyzed with Adobe Photoshop CS6 (Adobe, San Jose, CA, USA). The PBL was evaluated by measurement of the distance between the cement-enamel junction and the alveolar bone crest at 5 sites for each defect: the middle of the mesial root, the middle of the
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J Dent Res 93(2) 2014 185 Allogeneic BMMSC Transplantation for Periodontal Regeneration
Figure 2. Clinical and x-ray assessment of bone tissue regeneration. (A) Intra-oral photographs at 12 wks after transplantation, showing better bone tissue regeneration in the BMMSC+NaCl administration group than in the NaCl or untreated group. (B) Soft x-ray showed only limited bone formation in the untreated and NaCl groups, while alveolar bone regeneration was observed in the BMMSCs+NaCl group. (C-H) At 5 molar sites in 4 groups, probing bone loss (PBL) was lowest in the BMMSC+NaCl group compared with that in untreated and NaCl groups. (I) Bone regeneration rate (BRR) was calculated as [apex–cusp distance/apex–alveolar ridge distance]×100%. BRR was highest in the BMMSC+NaCl group compared with that in untreated and NaCl groups, indicating more bone tissue regeneration with BMMSC+NaCl injection than in the NaCl or untreated group. Statistical significance was evaluated by analysis of variance. All error bars represent SD (n = 10). *p < .05. **p < .01.
distal root, the middle of the middle root, the middle of the mesial furcation, and the middle of the distal furcation. All measurements at each site were made along the long axis of the root in the photograph, with scale tools provided by Photoshop (Fig. 1B). The PBL was calculated at the 5 sites for each molar, with a higher value representing more periodontal bone loss.
Measurement of Bone Regeneration Rate At 12 wks after injection, the bone regeneration rates (BRR) of the maxillary molars were examined by soft x-ray to measure the intrabony defects in 4 groups: healthy control, one group with no treatment, one group with injection of 0.9% NaCl, and
one group with injection of allogeneic BMMSCs+0.9% NaCl. The maxillary bone with the first molar was attached to a plastic board on top of an x-ray film (Kodak, Rochester, NY, USA). The specimen was placed perpendicular to the board to facilitate measurement of the height of the molar from the occlusal surface to the apical root and the alveolar ridge. The radiograph was exposed for 20 sec in an x-ray machine (Kyodo DMK 14z) at 30 kV and 2.5 mA and developed in an x-ray processor for 3 min. All measurements of the distal aspects of the first molar were performed with Photoshop software. The apex–cusp (AC) and apex–alveolar ridge (AR) distances were measured, and BRR was calculated as AR/AC×100% (Fig. 1C). Higher BRR values indicated less bone formation.
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Figure 3. HE staining showed new periodontal tissue regeneration in the periodontal defects of the BMMSC+NaCl group, while bone and periodontal ligament fiber shortages remained in the untreated and NaCl groups. The alveolar bone height in the BMMSC+NaCl group was also much larger than those in the untreated and NaCl groups. D, dentin; B, bone; PDL, periodontal ligament; CEJ, cemento-enamel junction; HAB, height of alveolar bone. Scale bar = 100 µm.
Statistical Analysis All statistical calculations were performed with SPSS13 statistical software. Statistical significance was determined by the independent sample test or analysis of variance. A p value ≤ .05 was considered significant.
Results Local Injection of Allogeneic BMMSCs Improved Tissue and Bone Regeneration in Periodontitis At 12 wks post-injection, intra-oral photographs showed that the BMMSC+NaCl injection group had better periodontal tissue regeneration than the NaCl and untreated groups, but less regeneration than the healthy controls (Fig. 2A). Quantitative analysis revealed the following PBL values at the mesial site: 0.84 ± 0.06 in the healthy group, 1.52 ± 0.06 in the BMMSC+NaCl injection group, 2.07 ± 0.18 in the NaCl injection group, and 2.18 ± 0.19 in the untreated group. The PBL values at the distal site were: 0.75 ± 0.05 in healthy group, 1.2 ± 0.15 in the BMMSC+NaCl injection group, 1.67 ± 0.09 in the NaCl injection group, and 1.68 ± 0.13 in the untreated group. PBL values at the mesial furcation site were: 0.79 ± 0.03 in the healthy group, 1.38 ± 0.04 in the BMMSC+NaCl injection group, 1.52 ± 0.12 in the NaCl injection group, and 1.63 ± 0.16 in the untreated group. At the distal furcation site, the PBL values were: 0.8 ± 0.04 in the healthy group, 1.06 ± 0.07 in the BMMSC+NaCl injection group, 1.39 ± 0.1 in the NaCl injection group, and 1.38 ± 0.09 in the untreated group. PBL values at the middle site measured: 0.84 ± 0.03 in the healthy group, 1.31 ± 0.13 in the BMMSC+NaCl injection group, 1.56 ± 0.14 in the NaCl injection group, and 1.68 ± 0.13 in the untreated group (Figs. 2C-2G). The average PBL values were 0.8 ± 0.21 in the healthy group, 1.2 ± 0.19 in the BMMSC+NaCl injection group, 1.6 ± 0.2 in the NaCl injection group, and 1.7 ± 0.14 in the untreated group (Fig. 2H). Statistical analysis indicated that, although local BMMSC injection could not restore tissues to healthy levels, it significantly
J Dent Res 93(2) 2014 improved periodontal tissue regeneration compared with NaCl injection or no treatment. Soft x-ray results showed more bone formation in the BMMSC+NaCl injection group compared with that in the NaCl and untreated groups, but formation did not reach normal levels (Fig. 2B). Quantitative analysis revealed that the BRR was 53% in the BMMSC+NaCl injection group, 45% in the NaCl injection group, and 44% in the untreated group (Fig. 2I), indicating that local BMMSC injection enhanced bone formation in periodontal defects. Histopathological results showed that the new bone and periodontal ligament in the periodontal defects in the BMMSC+NaCl group were regenerated to the highest levels compared with those in the NaCl and untreated groups. In contrast, the NaCl and untreated groups still showed typical periodontitis status, including deep periodontal pockets and shortage of new bone and periodontal ligament fibers (Fig. 3).
Local Injection of BMMSCs Inhibited the Expressions of TNFα, IFNγ, and IL1β The ELISA results showed that the concentration of TNFα was 2,674.88 ± 102.77 pg/mL in the BMMSC+NaCl group, 3,422.1 ± 51.98 pg/mL in the NaCl group, and 870.99 ± 28.02 pg/mL in the healthy group. IFNγ was 609.85 ± 25.5 pg/mL in the BMMSC+NaCl group, 803.79 ± 33.85 pg/mL in the NaCl group, and 212.88 ± 63.68 pg/mL in the healthy group; and IL1β was 1,038.46 ± 76.29 pg/mL in the BMMSC+NaCl group, 1,175.26 ± 105.55 pg/mL in the NaCl group, and 91.93 ± 38.14 pg/mL in the healthy group (Figs. 4A-4C). However, the concentration of IL6 was not significantly different (Fig.4D). The results showed that although TNFα, IFNγ, and IL1β could not be restored to healthy levels, their concentrations were significantly decreased in the periodontitis supernatants after BMMSC injection compared with those in the NaCl group, indicating an anti-inflammatory effect of local BMMSC injection in rat periodontitis.
Discussion Key features of periodontal disease include alveolar bone loss and gingival atrophy. For years, researchers have searched for methods to restore periodontal tissue and attachment. In previous studies, the delivery of stem cells to periodontal wounds involved both biomaterial-based and biomaterial-free approaches. Biomaterial systems included collagen sponges (Kawaguchi et al., 2004), gelatin (Kuo et al., 2008), or HA/TCP scaffolds (Liu et al., 2008) or similar matrices. Biomaterial-free methods involved cell sheet engineering (Wei et al., 2012) or cell pellets (Yang et al., 2009). The transplantation format depends on the surgical procedure. However, these treatments are suitable only for patients with severe periodontitis who are in a sufficient physical condition to tolerate surgery. Cell injection therapy is the most commonly used approach for treating a variety of diseases, including the regeneration of spinal cord injury (Koda et al., 2005) or myocardial infarction (Shake et al., 2002). Injected cells can be administered directly to defective tissues. The usefulness of BMMSCs can be attributed
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J Dent Res 93(2) 2014 187 Allogeneic BMMSC Transplantation for Periodontal Regeneration to their low immunogenicity and their immunomodulatory function. Thus, in the present study, we investigated the feasibility of BMMSC suspension injection in a rat periodontitis model. Initially, we observed lesion restoration in the untreated group, the NaCl injection group, and the BMMSC suspension group. According to clinical observation and radiographic and histological examination, we found that the BMMSC injection group contributed significantly more to periodontal tissue repair than did the untreated or the NaCl injection group. Although the main drawback of cell suspension injection is a high cell number loss and the unknown fate of BMMSCs in the local tissues, the results of this study demonstrated that the cells can work efficiently in the local tissue. In addition, animal experiments demonstrated that transplanted BMMSCs labeled with green fluorescent protein Figure 4. Local administration of BMMSCs inhibited TNFα, IFNγ, and IL1β expression. TNFα (GFP) were detectable by immunohisto(A), IFNγ (B), and IL1β (C) concentrations were significantly decreased in the periodontitis supernatants after BMMSCs+NaCl injection compared with those in the NaCl group. chemical analysis 4 wks after transplanStatistical significance was determined with the independent sample test. (D) No significant tation (Kawaguchi et al., 2004). differences of IL-6 were found among these groups. All error bars represent SD (n = 6). T-cell-mediated immunity is consid*p < .05. **p < .01. ered a critical factor in periodontitis pathogenesis. Gaffen and Hajishengallis System (2013-3-035 to Z.P.F.). The authors declare no potential (2008) described the Th (T-helper) 1/Th2 cytokine profile in conflicts of interest with respect to the authorship and/or publichronic periodontitis in humans. Our present results showed that cation of this article. the local injection of BMMSCs inhibited the expression of TNFα, IFNγ, and IL1β. These 3 cytokines play important roles in inflammation and immunity. IFNγ is produced by T-cells and enhances References T-cell-mediated immunity (Page et al., 1997). TNFα is reportedly Abramson SB, Yazici Y (2006). Biologics in development for rheumatoid involved in inflammatory response, bone loss, and autoimmune arthritis: relevance to osteoarthritis. Adv Drug Deliv Rev 58:212-225. diseases, including Crohn’s disease, ulcerative colitis, and multiAndreakos E (2003). Targeting cytokines in autoimmunity: new approaches, ple sclerosis (Andreakos, 2003; Abramson and Yazici, 2006). new promise. Expert Opin Biol Ther 3:435-447. Garlet et al. (2010) previously showed that inhibiting Treg funcBianco P, Riminucci M, Gronthos S, Robey PG (2001). Bone marrow strotion can increase IFNγ and TNFα levels and enhance alveolar mal stem cells: nature, biology, and potential applications. Stem Cells 19:180-192. bone loss and inflammatory cell migration. However, further Chang KM, Ramamurthy NS, McNamara TF, Evans RT, Klausen B, Murray investigations will be carried out to elucidate local immunological PA, et al. (1994). Tetracyclines inhibit Porphyromonas gingivalischanges in the periodontitis animal model. induced alveolar bone loss in rats by a non-antimicrobial mechanism. Taken together, our present results indicate that local injecJ Periodontal Res 29:242-249. tion of BMMSCs can repair tissue defects in cases of periodonFiorina P, Jurewicz M, Augello A, Vergani A, Dada S, La Rosa S, et al. (2009). Immunomodulatory function of bone marrow-derived mesentitis, as well as exert anti-inflammatory and immunomodulatory chymal stem cells in experimental autoimmune type 1 diabetes. functions in a rat model. This technique is promising as an easy J Immunol 183:993-1004. and non-invasive therapy for clinical periodontitis. Gaffen SL, Hajishengallis G (2008). A new inflammatory cytokine on the
Acknowledgments This work was supported by grants from the National Basic Research Program of China (No. 2010CB944801), the National Natural Science Foundation of China (81271101 to Z.P.F., 81070843 to Z.C.S.), the Program for New Century Excellent Talents in University (NCET-12-0611 to Z.P.F.), the Beijing Municipal Science and Technology Commission (Z1211000 05212004 to S.L.W.), and High-level Talents of Beijing Health
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