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The aim of this study is to validate a minimally invasive surgical procedure to harvest palate periosteum as a source of tissue for mesenchymal stromal/stem cells. We performed a standardized procedure to harvest the palate periosteum in ten subjects, which consisted of a 3 mm disposable punch and a Molt periosteal elevator to harvest a small full-thickness fragment of soft tissue at the hard palate area, between the upper bicuspids, 3 to 4 mm apical to the cement enamel junction. The one-third inner portion was fragmented, and following standard cell culture procedures, the adherent cells were cultured for three passages, after obtaining 70–90% confluence. Cell morphology analysis, flow cytometry analysis, and viability and osteogenic differentiation assays were performed. In all 10 cases, uneventful healing was observed, with no need for analgesic intake. The evaluation of cell morphology showed elongated spindle-shaped cells distributed in woven patterns. A high viability range was verified as well as an immunophenotype compatible with mesenchymal stem cell lineage. The differentiation assay showed the potential of the cells to differentiate into the osteogenic lineage. These results demonstrate that the minimally invasive proposed surgical technique is capable of supplying enough periosteum source tissue for stem cell culture and bone tissue engineering.

The aim of this study was to test whether lyophilized conditioned media from human dental pulp mesenchymal stem cell cultures promote the healing of critical-size defects created in the calvaria of rats. Prior to the surgical procedure, the medium in which dental pulp stem cells were cultured was frozen and lyophilized. After general anesthesia, an 8 mm diameter bone defect was created in the calvaria of twenty-four rats. The defects were filled with the following materials: xenograft alone (G1) or xenograft associated with lyophilized conditioned medium (G2). After 14 or 42 days, the animals were euthanized, and the specimens processed for histologic and immunohistochemical analysis. Bone formation at the center of the defect was observed only in the G2 at 42 days. At both timepoints, increased staining for VEGF, a marker for angiogenesis, was observed in G2. Consistent with this, at 14 days, G2 also had a higher number of blood vessels detected by immunostaining with an anti-CD34 antibody. In conclusion, conditioned media from human dental pulp mesenchymal stem cell cultures had a positive effect on the regenerative process in rat critical-size bone defects. Both the formation of bone and enhancement of vascularization were stimulated by the conditioned media.

The aim of this study was to histologically verify the performance of pulp-derived stem cells used in the pulp–dentin complex regeneration. Maxillary molars of 12 immunosuppressed rats were divided into two groups: the SC (stem cells) group, and the PBS (just standard phosphate-buffered saline) group. After pulpectomy and canal preparation, the teeth received the designated materials, and the cavities were sealed. After 12 weeks, the animals were euthanized, and the specimens underwent histological processing and qualitative evaluation of intracanal connective tissue, odontoblast-like cells, intracanal mineralized tissue, and periapical inflammatory infiltrate. Immunohistochemical evaluation was performed to detect dentin matrix protein 1 (DMP1). In the PBS group, an amorphous substance and remnants of mineralized tissue were observed throughout the canal, and abundant inflammatory cells were observed in the periapical region. In the SC group, an amorphous substance and remnants of mineralized tissue were observed throughout the canal; odontoblasts-like cells immunopositive for DMP1 and mineral plug were observed in the apical region of the canal; and a mild inflammatory infiltrate, intense vascularization, and neoformation of organized connective tissue were observed in the periapical region. In conclusion, the transplantation of human pulp stem cells promoted partial pulp tissue neoformation in adult rat molars.