1Department of Stem Cell Biology and Regenerative Medicine, National Center for Geriatrics and Gerontology, Obu, Japan

1Department of Stem Cell Biology and Regenerative Medicine, National Center for Geriatrics and Gerontology, Obu, Japan

1Department of Stem Cell Biology and Regenerative Medicine, National Center for Geriatrics and Gerontology, Obu, Japan

2Department of Endodontics, School of Dentistry, Aichi Gakuin University, Nagoya, Japan

3Innovation Center for Clinical Research, National Center for Geriatrics and Gerontology, Obu, Japan

4Department of Oral and Maxillofacial Radiology, School of Dentistry, Aichi Gakuin University, Nagoya, Japan

5Department of Dental and Oral Infrastructure Development, Center of Advanced Medicine for Dental and Oral Diseases, National Center for Geriatrics and Gerontology, Obu, Japan

Not applicable.

Experiments have previously demonstrated the therapeutic potential of mobilized dental pulp stem cells (MDPSCs) for complete pulp regeneration. The aim of the present pilot clinical study is to assess the safety, potential efficacy, and feasibility of autologous transplantation of MDPSCs in pulpectomized teeth.

Five patients with irreversible pulpitis were enrolled and monitored for up to 24 weeks following MDPSC transplantation. The MDPSCs were isolated from discarded teeth and expanded based on good manufacturing practice (GMP). The quality of the MDPSCs at passages 9 or 10 was ascertained by karyotype analyses. The MDPSCs were transplanted with granulocyte colony-stimulating factor (G-CSF) in atelocollagen into pulpectomized teeth.

The clinical and laboratory evaluations demonstrated no adverse events or toxicity. The electric pulp test (EPT) of the pulp at 4 weeks demonstrated a robust positive response. The signal intensity of magnetic resonance imaging (MRI) of the regenerated tissue in the root canal after 24 weeks was similar to that of normal dental pulp in the untreated control. Finally, cone beam computed tomography demonstrated functional dentin formation in three of the five patients.

Human MDPSCs are safe and efficacious for complete pulp regeneration in humans in this pilot clinical study.

Dental caries is a common health problem in humans. When dental caries is deep, reaching the dental pulp, the treatment of choice is generally pulpectomy. The dental pulp has several vital functions such as protection from infections by immunological surveillance, rapid reparative dentin formation to guard against noxious external stimuli, and maintenance of tensile strength to prevent tooth fractures [1]. Following pulpectomy and root canal filling, postoperative pain [2], apical periodontal lesions caused by microleakage from the tooth crown [3, 4], and vertical root fracture [5] may occur, leading to a higher incidence of extraction of the affected tooth. Recent advances in stem cell biology have aided stem cell therapy to regenerate the pulp/dentin complex for conservation and complete structural and functional restoration of the tooth by the triad of tissue engineering: 1) mesenchymal stem cells (MSCs), 2) growth/differentiation factors or cytokines, and migration/homing factors, and 3) the microenvironment (scaffold, extracellular matrix) [6]. We have demonstrated complete pulp regeneration by harnessing autologous dental pulp stem cell (DPSCs) subsets transplanted with stromal cell-derived factor 1 (SDF1) in a collagen scaffold into a canine pulpitis model [7, 8]. Next, a novel isolation method was developed employing an optimal granulocyte colony-stimulating factor (G-CSF)-induced mobilization of DPSCs for clinical-grade mesenchymal stem cells from a small amount of pulp tissue by good manufacturing practice (GMP)-grade guidelines [9]. G-CSF was already approved by the Food and Drug Administration (FDA) for clinical use. The isolated human mobilized DPSCs (MDPSCs) were characterized further by the higher migratory activity and trophic effects including migration, anti-apoptosis, and immunosuppression compared with colony-derived DPSCs in vitro. Furthermore, human MDPSCs demonstrated higher regeneration potential using an ectopic tooth root transplantation in severe combined immunodeficient (SCID) mice. Thus, MDPSCs have potential utility for pulp regeneration [9]. G-CSF was evaluated as an optimal GMP-grade migration/homing factor for pulp regeneration, having a variety of effects including anti-apoptosis on the transplanted and migrated cells, engraftment of the transplanted cells, angiogenesis, and immunosuppression [10]. The potential stem cell therapy for pulpitis harnessing MDPSCs with G-CSF was then examined in a preclinical study. Initially, the human MDPSCs isolated in a totally enclosed system in a GMP-compliant facility were evaluated by their karyotype, safety, and efficacy. Then, canine MDPSCs were isolated by the similar standard operating procedure (SOP) used in humans, and the preclinical feasibility, safety, and efficacy of pulp regeneration was established by autologous transplantation of the MDPSCs with GMP-grade G-CSF into the pulpectomized tooth in a canine pulpitis model [10]. On the basis of these preclinical safety and efficacy results and its mechanism for pulp regeneration, the protocol of a clinical study for pulp regenerative therapy was developed and approved by Institutional Review Boards and by the Japanese Ministry of Health, Labor and Welfare.

The aim of this investigation is to assess the safety, potential efficacy, and feasibility of autologous transplantation of human clinical-grade MDPSCs and to evaluate the utility of the stem cell therapy in a pilot clinical study for the first time. According to the Japanese guidelines of human stem cell clinical research, based on ethical considerations, only cases in which pulp tissue removal is inevitable should be selected for clinical study. In cases of severe irreversible pulpitis, including chronic ulcer pulpitis and acute suppurative pulpitis, the pulp tissue is exposed and the whole pulp tissue is infected, and there is no effective treatment other than whole pulp removal. Thus, we selected pulpectomized teeth due to severe irreversible pulpitis without periapical lesions for this purpose.

The pilot clinical study was conducted according to the principles of the Declaration of Helsinki and the Japanese guidelines of human stem cell clinical research, and to the standard of the manufacturing management and the quality control of pharmaceutical products and quasi-drug (Good Manufacturing Practice; GMP). Subjects were enrolled if they fulfilled the following inclusion criteria: aged between 20 and 55 years, diagnosis of irreversible pulpitis of single root canal, no fracture, a sound tooth structure remaining over the margin of the alveolar bone and no periapical radiolucency by X-ray analysis, and having a discarded tooth without deep caries to supply pulp tissue. Patients were excluded if they presented evidence of infection due to virus, bacteria, fungi and mycoplasma, severe cardiovascular disease, diabetes (HbA1c (NGSP) over 7.0%), osteoporosis, pregnancy, were mentally disabled or had mental disease. In addition patients who received antiplatelet agents or anticoagulant remedy and who had a history of allergy to antimicrobial and the local anesthetic agents and positive intracutaneous reaction for atelocollagen were excluded. Patients who could not receive magnetic resonance imaging (MRI) examination were also excluded. The enrolled patients for participation in the clinical study underwent autologous serum isolation and further extraction of a discarded tooth after signing informed consent again.

Autologous serum was isolated from freshly collected blood (200 ml) by Serum Collection Set (CELLAID®, JMS Co. Ltd., Hiroshima, Japan) in a GMP-compliant facility. The autologous discarded tooth was extracted, soaked in Hank’s balanced salt solution (Invitrogen, Carlsbad, CA, USA) after making a longitudinal cut, and transported to the GMP-compliant facility within 1 hour under strict temperature control at 0–10 °C (Testo, Yokohama, Japan). The isolation of MDPSCs was performed according to a standard operating procedure (SOP) under strict GMP conditions in a totally enclosed system of the Isolator (Panasonic Healthcare Co. Ltd., Tokyo, Japan) as described previously in the preclinical trial [10]. In brief, the pulp cells were isolated by enzymatic digestion in 0.04 mg/ml GMP-grade Liberase MTF (Roche, Mannheim, Germany) for 30 min at 37 °C, and were plated at 5.6–32.0 × 104 cells in a T25 flask (25 cm2; Sumitomo Bakelite Co. Ltd., Tokyo, Japan) in Dulbecco’s modified Eagle’s medium (DMEM; Sigma, St. Louis, MO, USA) supplemented with 10% autologous serum (autoserum), 2.5 mg/ml amphotericin B (Bristol-Myers Squibb, Tokyo, Japan), and 0.3% gentamicin (Nitten, Nagoya, Japan) which is only allowed in cell culture for clinical use in Japan and has low cytotoxicity. The scientific rationale for the use of autologous serum is to avoid any potential immune response/reaction to allogeneic and xenogeneic serum. DPSCs were detached by incubation with TrypLE™ Select (Invitrogen) before they attained 70% confluence. Mobilized DPSCs were further isolated by using a stem cell mobilization method under the previously determined optimal conditions: G-CSF (Neutrogin, Chugai Pharmaceutical Co. Ltd., Tokyo, Japan) at final concentration of 100 ng/ml, cell number 2 × 104 cells/100 μl on the Transwell (Corning, Lowell, MA) inserted into 24-well tissue culture plates with an incubation time of 48 h [9]. The isolated MDPSCs were further expanded at 1 × 104 cells/cm2 in DMEM (Sigma) supplemented with 10% autologous serum without antibiotics to passage 7 to obtain the required large number of MDPSCs for safety and quality control tests and 10-year cell cryopreservation according to the Japanese guideline of human stem cell clinical research as well as cell transplantation. They were cryopreserved at 1 × 106 cells/ml in a cryoprotectant, CP-1 (Kyokuto Pharmaceutical Industrial Co. Ltd., Tokyo, Japan), by gradually decreasing the temperature to –40 °C at the rate of –2 °C/min and further to –80 °C at the rate of –10 °C/min in a Programed Deep Freezer (Strex, Osaka, Japan). They were stored in a deep freezer (Sanyo Electric Co. Ltd, Osaka, Japan) at –80 °C until use.

The final cell product, MDPSCs at passage 7 of culture, was characterized by flow cytometry after immune-labeling with the antigen surface markers CD29, CD44, CD105, and CD31 as described previously [9]. The safety of MDPSCs during the process of tooth transportation, cell processing, cell freezing, and final transplantation was determined by sterility tests for fungi, aerobic and anaerobic bacteria, mycoplasma tests, endotoxin tests, and virus tests. In brief, the MDPSCs at passage 7 after cryopreservation and the MDPSCs combined with collagen and G-CSF used for transplantation in the operating room were sent independently to a quality-control referral laboratory (Tanabe R&D Service Co. Ltd., Saitama, Japan; SRL Inc., Tokyo, Japan; and BML Inc., Tokyo, Japan) for the tests. For the mycoplasma test, the real-time RT-PCR and DNA staining method were used according to the protocol (SRL Inc. and BML, Inc.). The cryopreserved MDPSCs were shipped for transplantation after confirming whether they meet the criteria of MSCs by a battery of in-process quality tests including cell surface marker analysis, cell viability, sterility, endotoxin, mycoplasma, and virus tests.

We examined chromosome aberrations, if any, in cell preparations at passages 9 or 10 of the culture stained with quinacrine mustard and Hoechst 33258 using a standard Q-banding procedure. Karyotypes were analyzed in metaphases of more than 20 cells in accordance with the Human Cytogenetic Nomenclature (ISCN) by entrustment (Chromosome Science Labo Inc., Sapporo, Japan).

Caries of the affected tooth was completely removed. In certain cases it was first necessary to supply a missing wall with composite resin (Clearfil DC core automix, Kuraray Noritake Dental Inc., Tokyo, Japan) with an adhesive procedure using a bonding agent (Clearfil Mega Bond, Kuraray Noritake Dental Inc.) (Fig. 1) to prevent the rubber dam clamp from slipping off the tooth as well as to isolate the root from the saliva and bacteria. The affected tooth was then pulpectomized. Apical shaping was performed to the cemento-dentinal junction or 0.5 mm under from the junction to the size of 0.45 to 0.55 mm after measuring the root canal length with a #25 K file using Root ZX (Morita Corp., Osaka, Japan). After that, the conventional root canal preparation was performed. Irrigation was carried out alternately with 6% NaOCl and 3% H2O2 and further with saline. An absorbent point moistened with minocycline (MINOMYCIN® IVD, Pfizer Japan Inc., Tokyo, Japan) or 0.5% levofloxacin (CRAVIT®, Santen Pharmaceutical Co. Ltd, Osaka, Japan) was carried into the root canal before cell transplantation as a conventional root canal treatment. The cavity was temporarily filled with a double-seal, water-setting hydraulic cement (Caviton; GC, Tokyo, Japan) and composite resin (Clearfil DC core automix) with an adhesive procedure (Clearfil Mega Bond). Water setting Caviton is advantageous to application of liquid antibiotics in the root canal (Fig. 1). For transplantation, the cryopreserved autologous MDPSCs at 1 × 106 cells were transported to the clean bench of the operating room, thawed, and suspended in 40 μl of a clinical-grade atelocollagen scaffold (Koken, Tokyo, Japan) and 300 ng of G-CSF (Neutrogin) after washing with saline. The root canal was dried well with paper points after irrigation with 3 ml each of 6% NaOCl and 3% H2O2 and 5 ml of saline, and further with 2 ml of 3% EDTA solution for 2 min (SmearClean, Nippon Shika Yakuhin Co. Ltd., Simonoseki, Japan) and 5 ml of saline. Half of the cell suspension (20 μl) was transplanted into the root canal by a cannula (indwelling needle, #26 gauge, Nipro, Osaka, Japan) paying close attention not to introduce any bubble inside. The Gelatin sponge (Spongel, Astellas Pharma Inc., Tokyo, Japan) was placed on the suspension in the root canal orifice without pressure, and the cavity was sealed with glass ionomer cement (GC Fuji IX EXTRA; GC, Tokyo, Japan) and composite resin (Clearfil DC core automix) with a bonding agent (Clearfil Mega Bond) (Fig. 1). The teeth were further covered with a hard resin jacket crown temporally with polycarboxylate temporary cement (Shofu Hy-Bond temporary cement hard, Shofu) in patients 1 and 3.

A sequence of illustrations describing step-by-step the sequences of the clinical study, including caries treatment with composite resin wall restoration followed by pulpectomy, cell processing, and cell transplantation, followed by final restoration. CBCT cone beam computed tomography, CPC Cell Processing Center, GMP good manufacturing practice, MDPSC mobilized dental pulp stem cell, MRI magnetic resonance imaging

The patients were followed up at 1, 2, 4, 12, and 24/28/32 weeks after MDPSC transplantation. For the safety evaluation, the incidence, severity, and outcome of immediate or delayed adverse events were recorded. As a first-in-human clinical pilot study under the Japanese guidelines of human stem cell clinical research, urine chemistry examinations and blood tests and blood chemistry examinations were performed at each visit except at 2 weeks. Twelve-lead electrocardiogram was monitored at 4 and 24 weeks. Local clinical examinations including percussion pain and tenderness at each visit and X-ray analyses for periapical lesion were also performed at the first visit (FV), pre-transplantation just before cell transplantation (Pre), and at 4, 12, and 24/28/32 weeks by two radiologists.

Efficacy assessment was performed by the pulp sensibility test using an electric pulp tester (VITALITY SCANNER; Yoshida Dental Trade Distribution Co. Ltd, Tokyo, Japan) at each visit by three dentists. Before electric pulp test (EPT), the surface of the tooth was dried well so as not to flow the current to the adjacent gingival or periodontal tissues. The probe tip was applied to the natural tooth structure, not to the restored part. Toothpaste was used for making good contact with the surface of tooth. The current was slowly increased to give accurate results. Another pulp sensibility test, the cold test, was performed using dicholorofluoromethane refrigerant spray (PULPER, GC Corp., Tokyo, Japan) at every visit. The frozen sponge was applied for a few seconds to the gingival third of the buccal part or any part of the dried tooth to give a good cold conduction. In addition, a 1.5 Tesla (T) MRI (Philips Electronics Japan, Tokyo, Japan) was used for imaging of regenerated tissue at baseline, and at 12 and 24 weeks post-transplantation. Axial fat suppression T2-weighted images (T2WI) were obtained with the use of the Turbo RARE T2 technique. The imaging parameters were: repetition time (TR) 2500 ms, echo time (TE) 70–80, DFOV 22 × 31.6 cm, AQM 336 × 428, average 4, 128 × 128 matrix, 0.234 × 0.234 cm pixel size, 3-mm slice thickness, and 10–20 slices FA 90, NEX 3, EC 1. MRI were analyzed by a computer-assisted manual segmentation (outlining) technique using OsiriX medical imaging software which is a fast DICOM viewer program for the Apple Macintosh (downloadable at www.osirix-viewer.com). The OsiriX program offers all the basic image manipulation functions of zoom, intensity adjustment and filtering with real-time performance. Relative signal intensity (SI) was expressed as the SI of regenerated tissue to SI of the surrounding dentin of the same tooth compared with SI of normal pulp to SI of the surrounding dentin in the opposite site. Relative SI was calculated in axial sections of apical and coronal parts of the root canal, respectively.

Evaluation of dentin formation along the dentinal wall at 16 and 28 weeks was performed by cone beam computed tomography (Alphard-3030, Asahi Roentgen Ind. Co. Ltd., Kyoto, Japan). Cone beam computed tomography images were analyzed using the OsiriX program. At least five measurements were made: the densities of the dental pulp, dentin formation, and dentin were 140–168, 448–525, and 996–1025, respectively. Therefore, the low-density area ranging from 0 to 425 was considered as the dental pulp. The areas with this density range were automatically deducted and the volumes of the dental pulp were calculated.

Data are reported as means ± SD. P values were calculated using Student’s t test and Tukey’s multiple comparison test method in SPSS 21.0 (IBM, Armonk, NY, USA).

Five patients with irreversible pulpitis were enrolled from May to December 2013 in this pilot clinical study. Baseline characteristics of each individual patient are depicted in Table 1. Three patients were men and two were women, aged 28.6 ± 10.0 years (range, 20–44 years). Four patients had chronic ulcer pulpitis and one had acute suppurative pulpitis at the time of enrollment. The transplantation of the MDPSCs was performed after 1 to 12 weeks following pulpectomy.

Baseline characteristics of the individual patients

a The positive reaction was detected by electric pulp test (EPT), indicating that the pulp tissue was alive on enrollment. The tooth was left with camphorated phenol application in the cavity for 3 months before root canal treatment, and the pulp tissue became completely necrosis with a periapical lesion at that time

Human primary DPSCs (Fig. 2a) formed a colony in 7–15 days (Fig. 2b), and clinical-grade human MDPSCs were further isolated utilizing G-CSF-induced stem cell mobilization in the isolator (Fig. 2c). The expanded MDPSCs were stellate with short processes or spindle in shape (Fig. 2d). Flow cytometry revealed that positive rates of CD29, CD44, CD105, and CD31 were 98.7 ± 1.2%, 99.5 ± 0.3%, 94.3 ± 7.9%, and 0.6 ± 0.4%, respectively. The mean total cell number at passage 7 of culture excluding patient 1 was 15.5 ± 4.0 × 106. After the thawing of the frozen cells at passage 7 the cell viability was 83.0 ± 6.7% (Table 2). There were no significant structural chromosomal abnormalities/aberrations in the karyotype of all diploid cells. However, there were a few chromosomal aberrations in patients 1 and 4 (Table 2). In patient 4, 45,X found in one out of 20 cells did not affect regeneration after cell transplantation, possibly due to the fact that the Y chromosome functions only during development. No structural abnormalities including irregular portion of chromosomal DNA and no more than two chromosomes of a pair (trisomy, tetrasomy) were observed. In patient 1, 45,X found in two out of 20 and 45,X,-9 was detected. However, further examination of 45 demonstrated no specific chromosome anomalies. Also, no structural abnormalities and no more than two chromosomes of a pair (trisomy, tetrasomy) were detected. Therefore, cells from patients 1 and 4 could be used safely for cell transplantation. MDPSCs showed no bacterial, fungal, mycoplasma, endotoxin, or virus contamination in the expanded cells at passage 7 of culture after cryopreservation and in the freeze-thawing cells combined with atelocollagen and G-CSF (Table 2).

Isolation of MDPSCs from an autologous discarded tooth. a Primary DPSCs forming a small colony on day 3. b The DPSCs on day 7. The colony increased in size. c MDPSCs at passage 2 of culture on day 3. d MDPSCs at passage 7 of culture on day 5 before cryopreservation

Cell biological characteristics, including viability, expression rate of stem cell markers, cell survival rate, and karyotype

a The mobilized dental pulp stem cells (MDPSCs) of patient 1 were damaged at passage 4 of culture due to the high temperature of the incubator since the air-conditioner was broken in the Cell Processing Center, and the cryopreserved MDPSCs at passage 3 were used for further expansion, resulting in a smaller number of cells at passage 7. Karyotype was analyzed at passage 10

b Excluding patient 1

No adverse events related to cell transplantation were observed by examination of blood and urine and twelve-lead electrocardiogram during 24 weeks of follow-up in all patients (Table 3). Clinical examinations demonstrated no postoperative pain, including percussion pain and tenderness, at all follow-up visits up to 24 weeks. The radiographic examinations made by two radiologists showed no significant changes in the periapical areas related to the cell therapy in three patients (patients 1, 3, and 5). The periapical lesion clearly diagnosed before transplantation was gradually reduced in size and radiolucency during 24 weeks follow-up. In patient 2 there was minor widening of periodontal ligament space at 24 weeks. There was widening of the periodontal ligament space at 12 weeks and periapical radiolucency at 24 weeks in patient 4 (Fig. 3a).

Safety tests of mobilized dental pulp stem cells at passage 7 of culture and at cell transplantation