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Department of Stem Cell Biology and Regenerative Medicine

Stem Cell Biology and Regenerative Medicine 

Cell Processing

★ Aims and Mission ★

We will contribute to rapid progress of regenerative dental medicine and regenerative medicine by promoting practical use of innovative techniques for tooth regeneration.

★ Summary of our research goals ★

  1. To promote innovative endodontic treatment using novel regenerative endodontics and to establish the department as a center for standardization and propagation of this therapy.
  2. To produce a super aging society in which the elderly population has good health through the functional survival and endurance of the tooth.
  3. To contribute to a super aging society in which elderly people live longer and healthy lives based on regenerative therapy
    for vascular and neurogenic diseases by dental pulp stem cells.

Head Statement by Misako Nakashima

◆ Research ◆

My research started with initial dentinogenesis and reparative dentin induction by partially purified bone morphogenetic proteins (BMPs). During stay in National Institute of Dental Research, NIH as a visiting scientist, I learned Cell Biology and Molecular Biology and studied on isolation of pulp progenitor cells and their differentiation into odontoblasts by BMPs (1992). After coming back from USA, I started to examine the role of recombinant BMP2 and 4 in reparative dentin formation in vivo (1994). After a short stay in Johns Hopkins University, I identified a novel member of BMP family, Gdf11 (1996), and a novel zinc finger transcription factor. GliH1. Furthermore, during stay in New York University (1997-1999), gene targeting of GliH1 was performed to analyze its function in tooth development. After that I have developed gene therapy and cell therapy with BMPs for dentin regeneration (2001-2003) with Dr. Iohara.  Furthermore, my research project was changed from dentin regeneration after pulpotomy into pulp regeneration after pulpectomy in 2003. We isolated and characterized of dental pulp stem cell (DPSC) subsets with high angiogenic/neurogenic potential (2008) and transplanted the DPSC subsets with SDF-1 or G-CSF into pulpectomized tooth to regenerate pulp tissue in dogs (2011).  We further demonstrated utility of DPSC subsets for total pulp regeneration by a preclinical study (2013).  This potential cell therapy was also examined in our recent clinical investigation, suggesting safety and efficacy for total pulp regeneration in clinic.

  1. Nakashima M.: Induction of dentin formation on canine amputated pulp by recombinant human bone morphogenetic proteins (BMP)-2 and -4. J Dent Res. 73(9): 1515-1522, 1994. PMID: 7929986
  2. Nakashima M., Reddi AH.: The application of bone morphogenetic proteins to dental tissue engineering. Nature Biotech. 21(9): 1025-1032, 2003. PMID: 12949568
  3. Iohara K., Zheng L., Ito M, Ishizaka R., Nakamura H., Into T, Matsushita K., : Regeneration of dental pulp after pulpotomy by transplantation of CD31-/CD146- side population cells from a canine tooth. Regen Med. 4(3): 377-385, 2009. PMID: 19438313
  4. Iohara K., Murakami M., Takeuchi N., Osako Y., Ito M., Ishizaka R., Utunomiya S., Nakamura H., Matsushita K., Nakashima M.:A novel combinatorial therapy with pulp stem cells and granulocyte colony-stimulating factor for total pulp regeneration. Stem Cells Transl. Med. 2(7): 521-533, 2013. PMID: 23761108

◆ Positions and Employment ◆

  • 1988-1998
    Assistant Professor, Dept. of Endodontics, Kyushu University
  • 1999-2005
    Assistant Professor, Dept. of Endodontics, Kyushu University
  • 2005-2010
    Chief, Dept. of Oral Disease Research, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology
  • 2009-2015
    Visiting Professor, Department of Endodontology, School of Dentistry, Aichi-gakuin University
  • 2011-2015
    Head, Dept. of Dental Regenerative Medicine, Center for Advanced Medicine for Dental and Oral Diseases,National Center for Geriatrics and Gerontology, Research Institute
  • 2011-present
    Visiting Professor, Dept. of Orthopedic Surgery, School of Medicine, UC Davis
  • 2013-present
    Visiting Professor, School of Dentistry, Tokushima University
  • 2015-present
    Head, Dept. of Stem Cell Biology and Regenerative Medicine, National Center for Geriatrics and Gerontology

◆ Other Experience and Professional Memberships ◆

  • 2016
    Vice President, Pulp Biology and Regeneration Group, IADR
  • 2017
    President, Pulp Biology and Regeneration Group, IADR

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Disadvantages of Ordinal Endodontic Treatment

Root canal treatment is limited success due to complicated morphology of root canals and not easy access to complete disinfection and artificial filling. Time consuming and difficult surgery may also decrease the success rate. In case of pulpectomy and primary root canal treatment, the success rate is 86% and 80%, respectively. As the root canal treatment is repeated, its success rate becomes lower and possibilities of tooth fracture and tooth loss increase.

Tooth Structure

Pulpectomy

 

 

 

Longevity of teeth

 

 

 

Significance of Pulp/Dentin Regeneration

Dental pulp has many important functions in the homeostasis and maintenance of teeth essential for their long life, leading to an individual’s good quality of life. Deep caries and pulp exposure have been treated by pulp capping or pulp amputation to preserve pulp tissue for vital therapy, with limited success.
The long-term goal of our research is to regenerate the dentin/pulp complex and restore its functions compromised by pulp injury and/or inflammation. The regenerative therapy for dentin/pulp is a promising approach for endodontic treatment to preserve tooth function, and to prevent secondary caries, and tooth fracture. Furthermore, it may contribute to longevity of tooth, leading to good health and longevity.

Pulp Dentin Reg.

 

 

Novel Safe & Efficacious Method to Isolate Clinical Grade Mobilized DPSCs

We have developed a safe and efficacious method for cell isolation to obtain clinical-grade human DPSCs from a small amount of pulp tissue. We devised a method utilizing G-CSF-induced stem cell mobilization, as stem cells have high migration potential. The insert membrane is chemically treated to avoid cell attachment. By G-CSF, some of colony-derived DPSCs plated on the membrane can migrate through the membrane, and these DPSC subsets are termed mobilized DPSCs (MDPSCs).The MDPSCs have high expression of angiogenic/neurotrophic factors, high trophic effects (anti-apoptosis, immunosuppression), and high migration potential.

Safe & Efficacious

 

 

Pulp/Dentin Regenerative Therapy by MDPSCs

The clinical grade MDPSC are isolated from autologous teeth and expanded according to a standard operating procedure (SOP) under strict Good Manufacturing Practices (GMP) conditions in a totally enclosed system of the Isolator for aseptic condition. The safety, efficacy and reproducibility of cell products and compliance with GMPs have to be ensured. Every manufacturing process is recorded for quality control by computerized process management system of the Isolator, preventing and minimizing human error. After confirming the safety and quality, the cryopreserved MDPSCs are transported to an operating room and mixed with the drug approved migration factor, G-CSF and atelocollagen. The mixture is transplanted into the root canal after conventional root canal treatment.
Our preclinical study has demonstrated that the pulp-like tissue with well-vascularization and re-innervation is regenerated to the cementum-enamel junction 60 days after autologous transplantation of MDPSCs with G-CSF, indicating coverage of more than 90% of the total area of the root canal. Dentin formation is also observed along the dentinal wall.

This regenerative endodontics is potent ideal treatment to recover tooth function, and prevent secondary caries, re-treatment and tooth fracture, leading to longevity of tooth.

How to Regenerate

 

 

Clinical Success in Human Pulp Regenerative Therapy

We have performed a pilot clinical study of pulp regeneration after getting approval by Japanese Ministry of Health and Welfare. Primary purpose was to confirm safety, and secondary purpose was to evaluate efficacy of the regenerative pulp therapy. Applicable patients were suffering from irreversible pulpitis and were limited to those having their own discarded tooth. Five patients were registered after clinical study agreement. After registration, autologous serum was isolated, and autologous tooth was extracted for MDPSC isolation. Clinical examinations, oral examination, and X-ray, MRI and cone beam computed tomography (CBCT) were sequentially performed after transplantation up to at least 24 weeks.
There were no structural chromosomal abnormalities/aberrations in the karyotype of 46 diploid in every cell product, although there were a few numerical chromosomal aberration in two patients, 45, X. MDPSCs showed no bacterial, fungal, mycoplasma, endotoxin or virus contamination.
No adverse events related to cell transplantation were observed by blood test, urine test and electrocardiogram at all follow-up visits up to 24 weeks in any patients. Local clinical examinations demonstrated little postoperative pain including percussion pain and tenderness at each visit. The radiographic diagnosis showed no significant changes in the periapical areas related to the cell therapy in three patients. In one patient, the periapical lesion clearly diagnosed before transplantation was gradually reduced in size, and a little radiographic periodontal ligament space widening was observed at 24 weeks. In another patient, however, the widening of periodontal ligament space without any symptom was revealed at 24 weeks.
Assessment of pulp vitality by electric pulp test (EPT) demonstrated a positive response within 4 weeks in four patients, suggesting functional re-innervation in the root canal. One patient, however, showed no positive response during 24 weeks, since there had been already periapical radiolucency at the time of cell transplantation although the pulp tissue was alive at enrollment. The negative vital reaction was finally changed into positive after 36 weeks. Efficacy evaluation was also performed by magnetic resonance imaging (MRI). The signal intensity (SI) of the root canal was expressed in comparison with that of the surrounding dentin, and the relative signal intensity was further expressed as the affected tooth to normal tooth. The SI at 24 weeks became similar to that of normal pulp, suggesting that pulp tissue may be regenerated in the four patients. The radiographic interpretation on CBCT at 28 weeks demonstrated lateral dentin formation in three patients.

 These results suggested that MDPSC transplantation might be safe and efficacious for total pulp regeneration in humans. Further studies to enhance dentin formation in the crown part and root canal disinfection are needed to confirm the clinical protocol.

Patients

 

 

Successful Pulp and Periapical Tissue Regeneration in case of Periapical Disease

In the canine periapical disease model, disinfection of the root canal and MDPSC transplantation were performed by the same protocol as that in pulpectomized tooth. The pulp tissue and periapical tissue were regenerated. Thus, this regenerative pulp therapy may also be applied for periapical disease.

 

Periapical Disease Model

 

 

Other Tissue Regeneration by autologous or allogeneic DPSCs

MDPSCs express pluripotent stem cell markers, such as Oct4, Nanog, and Sox2 and angiogenic/neurotrophic factors. MDPSC secretome have more angiogenic, neurogenic potential and more migratory effect than bone marrow stem cell (BMSC) secretome.

 These properties actually make them a good candidate for the treatment of ischemic diseases and neurodegenerative diseases. We have demonstrated that MDPSCs are capable of promoting peripheral nerve regeneration in sciatic nerve defect model, central nerve regeneration in ischemic brain injury model, and vasculogenesis/angiogenesis in ischemic hindlimb. Moreover, we have recently evaluated the therapeutic potential of MDPSC secretome for Alzheimer's disease. The neuroprotective effects of MDPSC secretome including the Amyloid beta (Aβ)-degrading enzyme, neprilysin (NEP) and anti-apoptotic factors render MDPSCs promising candidates for secretome-based therapy for Alzheimer's disease.

Stem Cell Banking

 

 

 

 

 

 

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Achievements of Paper

◆ 2017 ◆

  1. Nakayama H., Iohara K., Hayashi Y., Okuwa Y., Kurita K., Nakashima M.: Enhanced regeneration potential of mobilized dental pulp stem cells from immature teeth. Oral Dis. Jul; 23(5):620-628. 2017. DOI: 10.1111/odi.12619.
  2. M., Iohara K., Murakami M., Nakamura H., Sato Y., Ariji Y., Matsushita K.: Pulp regeneration by transplantation of dental pulp stem cells in pulpitis: A pilot clinical study. Stem Cell Res Therapy Mar 9; 8(1):61, 2017. DOI: 10.1186/s13287-017-0506-5.

◆ 2016 ◆

  1. Yamamoto T., Osako Y., Ito M., Murakami M., Hayashi Y., Horibe H., Iohara K., Takeuchi N., Okui N., Hirata H., Nakayama H., Kurita K., Nakashima M.: Trophic effects of dental pulp stem cells on schwann cells in peripheral nerve regeneration. Cell Transplant. 25(1), 183-193, 2016.
  2. Hirose Y., Yamamoto T., Nakashima M., Funahashi Y., Matsukawa Y., Yamaguchi M., Kawabata S., Gotoh M.: Injection of dental pulp stem cells promotes healing of damaged bladder tissue in a rat model of chemically induced cystitis. Cell Transplant. 25(3), 425-436, 2016.
  3. Iohara K., Fujita M., Ariji Y., Yoshikawa M., Watanabe H., Takashima A., Nakashima M.: Assessment of pulp regeneration induced by stem cell therapy by Magnetic Resonance Imaging (MRI). J. Endod. 42(3):397-401, 2016.
  4. Kawamura R., Hayashi Y., Murakami H, Nakashima M.: EDTA soluble chemical components and the conditioned medium from mobilized dental pulp stem cells (MDPSCs) contain inductive microenvironment, promoting cell proliferation, migration and odontoblastic differentiation. Stem Cell Res. Ther. 7(1):77, 2016.
  5. Hirose Y., Yamaguchi M., Kawabata S., Murakami M., Nakashima M., Gotoh M., Yamamoto T.: Effects of extracellular pH on dental pulp cells in vitro. J Endod. 42(5): 735-741, 2016.
  6. Ahmed NE., Murakami M., Hirose Y., Nakashima M.: Therapeutic potential of dental pulp stem cell secretome for Alzheimer’s disease treatment: an in vitro study. Stem Cell International. Article ID 8102478, Volume 2016.

◆ 2015 ◆

  1. Takeuchi N., Hayashi Y., Murakami M., Alvarez F. J., Horibe H., Iohara K., Nakata K., Nakashima M.: Similar in vitro effects and pulp regeneration in ectopic tooth transplantation by basic fibroblast growth factor and granulocyte-colony stimulating factor. Oral Dis. 21(1):113-122, 2015.
  2. Murakami M., Hayashi Y., Iohara K., Osako Y., Hirose Y., Nakashima M.: Trophic effects and regenerative potential of mobilized mesenchymal stem cells from bone marrow and adipose tissue as alternative cell sources for pulp/dentin regeneration. Cell Transplantation 24(9):1753-1765, 2015.
  3. Kobayashi Y., Nakashima M., Wakakuri T., Imaki J., Ito M.: Histology and immunohistochemistry of the developing juxta-oral organ in mice. Ann. Anat. 198, 49–57, 2015.
  4. Hayashi Y., Murakami M., Kawamura R., Ishizaka R., Fukuta O., Nakashima M.: CXCL14 and MCP1 are potent trophic factors associated with cell migration and angiogenesis leading to higher regenerative potential of dental pulp side population cells. Stem Cell Res. Ther. 6:111, 2015. Erratum in. Stem Cell Res Ther. 7(1):86, 2016.

◆ 2014 ◆

  1. Albuquerque M.T.P., Valera M.C., Nakashima M., Nör J.E., Bottino M.C.: Tissue engineering-based strategies for regenerative endodontics. J. Dent. Res. 93(12), 1222-1231, 2014.
  2. Nakashima M., Iohara K.: Mobilized dental pulp stem cells for pulp regeneration: initiation of clinical trial. J Endod. 40(4 Suppl):S26-32, 2014.
  3. Fijita M., Iohara K., Horiba N., Tachibana K., Nakamura H., Nakashima M.: Pulp regeneration after complete disinfection of the root canal system by enhanced delivery of medicaments using ultrasound with nanobubbles in a canine periapical disease model. Jpn. J. Conserv. Dent. 57(2): 170-179,2014.
  4. Iohara K., Murakami M., Nakata K., Nakashima M.: Age-dependent decline in dental pulp regeneration after pulpectomy in dogs. Exp. Gerontol. 52:39-45, 2014. Erratum in. Exp Gerontol. 76:89, 2016.
  5. Hagiwara M., Kokubu E., Sugiura S., Komatsu T., Tada H., Isoda R., Tanigawa N., Kato Y., Ishida N., Kobayashi K., Nakashima M., Ishihara K., Matsushita K.: Vinculin and Rab5 complex is requited for uptake of Staphyrococcus aureus and interleukin-6 expression. PLOS ONE. 9(1):e87373, 2014.
  6. Takimoto K., Kawashima N., Suzuki N., Koizumi Y., Yamamoto M., Nakashima M., Suda H.: Down-regulation of Inflammatory Mediator Synthesis and Infiltration of Inflammatory Cells by MMP-3 in Experimentally-induced Rat Pulpitis. J. Endod. Sep; 40(9):1404-1409, 2014.
  7. Horibe H., Murakami M., Iohara K., Hayashi Y., Takeuchi N., Takei Y., Kurita K., Nakashima M.: Isolation of a stable subpopulation of mobilized dental pulp stem cells (MDPSCs) with high proliferation, migration, and regeneration potential is independent of age. PLoS One. 9(5): e98553, 2014. Erratum in. PLoS One. 11(3). e0151741, 2016.
  8. Miyashita S., Ahmed N.E., Murakami M., Iohara K., Yamamoto T., Horibe H., Kurita K., Takano-Yamamoto T., Nakashima M.: Mechanical forces induce odontoblastic differentiation of mesenchymal stem cells on three-dimensional biomimetic scaffolds. J Tissue Eng. Regen. Med. 2014 Jun 12. doi: 10.1002/term.1928.
  9. Hirose Y., Murakami M., Hayashi Y., Osako Y., Yamamoto T., Gotoh M., Nakashima M.: Augmentation of regenerative potential of mesenchymal stem cells by granulocyte-colony stimulating factor (G-CSF) induced mobilization. J. Stem Cell Res. Transplant. 1(2): 1006, 2014.

◆ 2014 ◆

  1. Ishizaka R., Hayashi Y., Iohara K., Sugiyama M., Murakami M., Yamamoto T., Fukuta O., Nakashima M.: Stimulation of angiogenesis, neurogenesis and regeneration by side population cells from dental pulp. Biomaterials. 34(8): 1888-1897, 2013.
  2. Iohara K., Murakami M., Takeuchi N., Osako Y., Ito M., Ishizaka R., Utunomiya S., Nakamura H., Matsushita K., Nakashima M.: A novel combinatorial therapy with pulp stem cells and granulocyte colony-stimulating factor for total pulp regeneration. Stem Cells Transl. Med. 2(7): 521-533, 2013. 
  3. Iohara K., Murakami M., Takei Y., Horibe H., Kurita K., Nakashima M.: Quality assurance of clinical-grade pulp stem cells manufactured in GMP-compliant facility. Jpn. J. Conserv. Dent. 56(2):121-129, 2013.
  4. Murakami M., Horibe H., Iohara K., Hayashi Y., Osako Y., Takei Y., Nakata K., Motoyama N., Kurita K., Nakashima M.: The use of granulocyte-colony stimulating factor induced mobilization for isolation of dental pulp stem cells with high regenerative potential. Biomaterials. 34(36): 9036-9047, 2013. Erratum in: Biomaterials. 166-167, 2016.
  5. Nakashima M., Iohara K., Murakami M.: Dental pulp stem cells and regeneration. Pulp Regeneration. Endodontics Topics. 38-50, 2013.

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Awards

◆ 2017 ◆

  1. Iohara K., Fujita M., Ariji Y., Yoshikawa M., Watanabe H., Takashima A., Nakashima M.
    Assessment of pulp regeneration induced by stem cell therapy by Magnetic Resonance Imaging (MRI). The 2017 Journal of Endodontics Awards Apr. 26, 2017.

◆ 2014 ◆

  1. Nakashima M.
    International Association for Dental Research Distinguished Scientist Award for Pulp Biology & Regeneration (IADR). June 25, 2014.
     

◆ 2013 ◆

  1. Iohara K., Takeuchi N., Nakamura H., Nakashima M.

    A novel combinatorial therapy with pulp stem cells and G-CSF for total pulp regeneration.International Federation of Endodontic Association, The 9th World Endodontic Congress, Best Presentation Award.May 26, 2013.

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Pamplet

Regenerative Therapy Pamphlet

The Pamphlet for Pulp Regenerative Therapy

(PDF:2363KB)PDFファイル

 

 

 

 

 

 

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NCGG

  • National Center for Geriatrics and Gerontology
  • National Hospital for Geriatric Medicine, NCGG
  • Center for Development of Advanced Medicine for Dementia
  • Center for Gerontology and Social Science

National Center for Geriatrics and Gerontology

7-430 Morioka-cho, Obu City, Aichi Prefecture, Japan