Transplantation of Allogeneic Mesenchymal Stromal Cells in Patients With Immature Apex Teeth With Pulp Necrosis and Chronic Apical Periodontitis

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).