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.

Background: It is known that a large number of mediators involved in osteogenesis can influence bone development and repair; however, whether these mediators could be used as markers of bone maturity has yet to be determined. Aim: To evaluate the expression of osteocalcin (OC) and Runt-related transcription factor 2 (Runx2) in bone biopsies obtained during the reconstruction of atrophic anterior maxillae using particulate bone xenografts with or without association of autogenous bone marrow aspirate concentrate (BMAC). Materials and Methods: Ten patients were distributed into two groups (n = 5), according to the type of grafting material used: Control group (CG), particulate bone xenograft alone, and test group (TG), particulate bone xenograft combined with BMAC. A bone specimen was removed from the graft area 4 months after grafting, before implant placement. The specimens were processed and submitted to immunohistochemical analysis for detection of OC and Runx2. Histomorphometry was used to ascertain the percentage of stained areas in both groups. The Wilcoxon Mann–Whitney U-Test was used in the statistical analysis (P < 0.05). Results: The immunohistochemical analysis revealed a significantly higher OC expression in the TG than in the CG, namely 27.40 ± 1.34% and 11.40 ± 2.70%, respectively (P < 0.05), and a significantly higher Runx2 expression in the TG than in the CG, namely 2.80 ± 0.84% and 0.40 ± 0.55%, respectively (P < 0.05). Conclusion: The OC and Runx2 expression levels were higher when BMAC was associated with the bone xenograft than when it was not.

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.

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.

Periosteum-derived osteo-like cells (PDOCs) and their lyophilized conditioned medium are promising tools for bone regeneration, but their therapeutic potential for osteoporosis remains unknown. This study evaluated the effects of PDOCs and their lyophilized conditioned medium on bone mineralization and microarchitecture in ovariectomized rats. Eighteen ovariectomized rats (six animals per group) received PBS (negative control group—NCG), PDOCs (periosteum osteo-like cell group—POCG), or a PDOC lyophilized conditioned medium (periosteum osteo-like lyophilized conditioned medium Group—POCM). All animals were euthanized after 42 days, and the femurs were submitted to microCT and histomorphometric analysis to determine: (1) thickness of the cortical bone; (2) number of trabeculae; (3) percentage of mineralized tissue; (4) percentage of collagen fibers; and (5) percentage of medullary spaces. The thickness of the cortical bone was statistically higher in POCG and POCM than in NCG animals (p < 0.05). The trabecular number was higher in POCG and POCM than in NCG rats (p < 0.05). The percentage of mineralized tissue was higher in POCG and POCM than in NCG animals (p < 0.05). No statistically significant difference was observed for collagen fibers or percentage of medullary spaces among groups. The use of PDOCs and their lyophilized conditioned medium improved cortical bone thickness, the number of trabeculae, and the level of mineralized tissue, suggesting that tissue engineering approaches with PDOCs and their lyophilized conditioned medium have potential use for the treatment of osteoporosis.

Periosteum-derived osteo-like cells (PDOCs) and their lyophilized conditioned medium are promising tools for bone regeneration, but their therapeutic potential for osteoporosis remains unknown. This study evaluated the effects of PDOCs and their lyophilized conditioned medium on bone mineralization and microarchitecture in ovariectomized rats. Eighteen ovariectomized rats (6 animals per group) received PBS (Negative Control Group–NCG); PDOCs (Periosteum Osteolike Cell Group - POCG), or PDOCs lyophilized conditioned medium (Periosteum Osteolike lyophilized conditioned medium Group - POCM). All animals were euthanized after 42 days, and the femurs submitted to microCT and histomorphometric analysis to determine: (1) thickness of the cortical bone; (2) number of trabeculae; (3) percentage of mineralized tissue; (4) percentage of collagen fibers; and (5) percentage of medullary spaces. The thickness of the cortical bone was statistically higher in POCG and POCM than in NCG animals (p<0.05). The trabecular number was higher in POCG and POCM than in NCG rats (p<0.05). The percentage of mineralized tissue was higher in POCG and POCM than in NCG animals (p<0.05), and no statistically significant difference was observed for collagen fibers and percentage of medullary spaces among groups. The use of PDOCs and their lyophilized conditioned medium improved cortical bone thickness, the number of trabeculae and the level of mineralized tissue, suggesting that tissue engineering approaches with PDOC and their lyophilized conditioned medium have potential use for the treatment of osteoporosis.