Transplantation of Allogeneic MSC in Patients With Pulp Necrosis and Chronic Apical Periodontitis
Condition: Pulp Necroses; Apical Periodontitis
MeSH terms: Periodontitis; Periapical Periodontitis; Dental Pulp Necrosis; Necrosis
Study type: Interventional
Phase: Phase 2/Phase 3

Summary: 
      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.
    

Detailed description: 
      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.
    

Intervention name(s): Allogenic transplant of BM-MSC in a root canal from patients with immature apexes and pulpar necrosis and apical periodontitis
Intervention type(s): Biological
Intervention description(s): The implant of BM-MSC/MSC-Endo/PRP in a clean and shaped root canal from patients with immature apexes and pulpal necroses and apical periodontitis
Intervention model: Single Group Assignment
Intervention model description: PRP clot loaded of BM-MSC in order to regenerate dental pulp tissue, placed in the root canal of an immature tooth from a patient with pulp necrosis and chronic apical periodontitis
Masking: None (Open Label)
Primary purpose: Treatment
Keywords: BM-MSC; Pulp regeneration