The purpose of the study is to evaluate the effect of Mesenchymal Stromal Cell (MSC) implantation on pulp and periapical regeneration of immature teeth with pulp necrosis and chronic apical periodontitis.

BACKGROUND:

• Post-traumatic pulp necrosis prevents root development in children and adolescents.
• The multipotent ability of MSC to differentiate into bone-forming cells (osteoblasts) and dentin-forming cells (Odontoblast) has allowed the development of protocols to induce dental pulp regeneration in preclinical models and patients with immature teeth with pulpal necrosis.

IMPACT:

• Worldwide, post-traumatic pulp necrosis in children and adolescents constitutes a health problem in the endodontic area.
• Treatment with MSC would provide an effective therapeutic alternative to patients with pulp necrosis and incomplete root formation.
• The possible pulp and periapical regeneration of immature teeth induced by MSC would have a huge impact on the treatment of these patients.

Eligibility for EMC implant study Age: 6 to 16 years Sex: Male or Female Healthy volunteers accepted: NO.

TREATMENT GROUPS:

In the present study, the implantation of MSC will be performed in patients with immature teeth with pulpal necrosis with apical periodontitis, who will receive the appropriate endodontic treatment (according to the guidelines of the American Association of Endodontics) and implantation of allogeneic BM-MSC . This group will be compared with the history made in the Postgraduate Endodontics of the Universidad Central de Venezuela (UCV) and with international case series made by revascularization.

Clinical follow-up of each patient:

1. Clinical controls (facial evaluation, gingival evaluation, apical palpation, horizontal and vertical percussion, cold and heat sensitivity tests) will be carried out on days 0, 7, 30, 90, 180 and 364. Additionally, a clinical evaluation will be carried out at the two years post-implantation of MSC.
2. Radiological controls will be carried out on days 0, 7, 30, 90, 180 and 364. Additionally, they will be carried out two years post-implantation of MSC.
3. A tomographic evaluation will be performed when was evident periapical repair in a periapical radiograph. To measure root formation, root canal narrowing and verification the periapical repair in 3D.

The endodontic procedure in the patients included in this study will focus on cases of pulp necrosis with apical periodontitis without evidence of infectious processes.

MATERIALS AND METHODS. Reagents Murine monoclonal antibodies, directed against human differentiation antigens (CD34, CD45, CD14, CD90, CD73, CD29, CD49b, CD166), conjugated to fluorescein isothiocyanate (CFI) or phycoerythrin (PE) were purchased from BD Biosciences (USA).

Isolation and culture of mesenchymal stromal cells (MSC) obtained from human bone marrow. In the present study, isolated EMFs from bone marrow (BM) from patients with a diagnosis of post-traumatic nonunion (failure of a bone union at fracture sites) will be used. These cells were transplanted into the pseudoarthrosis site to induce bone regeneration in these patients. The protocol for bone regeneration through EMF transplantation was carried out at the University Hospital of Caracas, Hospital Universitario de Los Andes, Hospital Pérez de León II and has the approval of the Bioethics Committees of each institution and each patient through informed consent. In this protocol, the BM of each patient was isolated by a puncture in the iliac crest. This procedure was performed in the operating room, under anesthesia and by a medical specialist. The MO aspirate was placed in alpha-MEM medium (Invitrogen, USA) with heparin (Sanofi Aventis). The mononuclear cells were separated by centrifugation on a Ficoll-Hypaque gradient (GE Healthcare, Sweden) and cultured in alpha-MEM-Chang medium (Irvine Scientific, USA) enriched with 20% autologous serum. These cells were kept in culture in a controlled environment at 37ºC and 5% CO2. After 72 hours, non-adherent cells were eliminated, and a basal culture medium (alpha-MEM-Chang / 20% autologous serum) was added. Their adherence to the plastic isolated the MSCs. Culture medium exchanges were made until reaching a confluence close to 70-80%. The MSCs were expanded by pealing the cultures, following the process described above. Microbiological examinations were performed after obtaining the BM and before performing the MSC implantation. After using MSC, a batch of these cells were cryopreserved at -70 -C.

Phenotypic characterization of MSC. Phenotypic characterization studies of MSC were carried out by flow cytometry. For which the MO adherent cells were detached from the culture flask by using trypsin-like enzymes. Subsequently, the cells were incubated with antibodies specific for MSC markers (CD90, CD73, CD105, CD29, CD166 and CD49b) and hematopoietic (CD34, CD45 and CD14). Cytometric analysis of the expression markers showed that 100% of the cells used for transplantation in each patient were MSC.

MSC differentiation studies. The multipotential differentiation capacity of MSCs was examined by culturing these cells in osteogenic, chondrogenic and adipogenic differentiation media, following a methodology similar to that previously described. Briefly, the MSCs were detached and seeded in 24-well culture plates at a cell density of 5x104 per well, proceeding to add the corresponding differentiation medium. For osteogenic differentiation, MSCs were cultured in the presence of basal medium enriched with dexamethasone (100nM, Biotech), ascorbic acid (10mM, Sigma), inorganic phosphate (1.8mM, Merck) and beta-glycerol phosphate (2mM, Sigma). For chondrogenic differentiation, cells were cultured in a commercial medium for chondrocytes (Cell Application, USA) and for differentiation towards the adipogenic lineage the commercial medium NH Adipodiff Human (Miltenyi, USA) was used. In all cases, the cells were kept in culture for 21-28 days with medium changes every 4-5 days. To demonstrate the changes associated with the differentiation process, the cells were fixed using paraformaldehyde (Merck, USA) and specific stains were performed for each case. Briefly, alizarin red to detect calcium deposition (evidence of osteogenesis), Alcian blue to detect proteoglycans (evidence of chondrogenesis), and oil red (Oil Red) to see lipids (evidence of adipogenesis). In all cases, microscopic observation and photographic registration were carried out. For endothelial differentiation (CEn), EMFs were cultured in MCDB 131 medium (Invitrogen, USA) enriched with 10% autologous serum, 10µg / ml of human epidermal growth factor (hu-EGF, R&D) and hydrocortisone (1µg / ml, Sigma).

MSC implantation in patients with pulp necrosis and apical periodontitis. All MSC processing procedures will be carried out in the cleanroom of the IVIC Cell Therapy Unit following the standards of good manufacturing practice (GMP). Allogeneic BM-MSC from patients diagnosed with post-traumatic nonunion and treated with implantation of these cells will be used to induce bone regeneration. The MSC to be used in this protocol have previously been phenotypically and functionally characterized. The MSC will be thawed, grown and expanded as previously described. A part of the cells will be kept in a medium for MSCs, and another part will be cultured in endothelial differentiation medium (CEM-Endo). Once the required number of MSCs and MSCs-Endo have been reached, a suspension of these cells (75,000 cells from each of them) will be placed in sterile culture tubes containing DMEM-F12 culture medium, without phenol red, supplemented with 20% autologous serum. Each tube containing MSC / MSC-Endo will be transported in a small biological sample transport cellar, at room temperature, to the Dentistry Service of the Instituto Venezolano de Investigaciones Científicas (IVIC).

Under sterile conditions, the patient will be locally anesthetized in the affected tooth area; the root canal of the affected tooth will be exposed and prepared to perform the MSC / MSC-Endo / PRP implant. At the same time, the culture medium supernatant is removed from each tube and the CEM / CEM-Endo "button (pellet)" is resuspended in autologous platelet-rich plasma (PRP). Subsequently to the MSC / Endo / PRP suspension, 5% CaCl2 and thrombin will be added. Immediately, and before the clot forms, 20 microliters of the CEM / CEM-Endo / PRP suspension will be placed in the root canal, covered with a collagen membrane. Subsequently, the obturation procedure with bioceramics will be carried out at the level of the pulp chamber, ionomeric glass to protect the bioceramic and later composite resin to restore the tooth.

Post-implantation clinical evaluation of EMF

1. Clinical controls (facial evaluation, gingival evaluation, apical palpation, horizontal and vertical percussion, cold and heat sensitivity tests) will be carried out on days 0, 7, 30, 90, 180 and 364. Additionally, a clinical evaluation will be carried out at the two years post-implantation of EMC.
2. Radiological controls will be carried out on days 0, 7, 30, 90, 180 and 364. Additionally, they will be carried out two years post-implantation of mesenchymal stromal cells.
3. A tomographic evaluation will be performed when the periapical repair will be evident in a periapical radiograph.

Inclusion Criteria:

• Diagnosis of pulp necrosis and apical periodontitis in teeth with immature apices.
• Informed consent by the patient's representative and consent by the patient to receive bone marrow allogeneic mesenchymal stromal cell transplantation treatment.

Exclusion Criteria:

• HIV positive
• Hepatitits B or C positive
• Autoimmune diseases: lupus, rheumatoid arthritis.
• Neoplastic diseases.
• Major metabolic disorders
• Pregnancy
• Being on steroid treatment
• Other criteria that the researchers consider inappropriate for the inclusion of the patient

Possibly we can share the individual clinical, radiograph and tomographic outcome of every patient enrolled in the study, mainly focus on the difficulty that every patient present.