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: Osteoporosis is a systemic disease characterized by a progressive decrease in bone density and deterioration of the tissue’s microarchitecture. This results in greater structural fragility and a higher risk of fractures. Osteopenia represents the beginning of the process of decreasing bone density and, if left untreated, can lead to osteoporosis. The objective of this study was to validate an experimental model for establishing cases of decreased bone density that allows for the creation of different levels of severity of mineral loss and changes in bone microstructure. Materials and Methods: Twenty female Wistar rats, 12 weeks old and with a body weight ranging from 300 to 400 g, were used in this study. The animals were randomly distributed into five groups (n = 5 per group): a control group (CG), where the animals were not ovariectomized (OVX), and four experimental groups, where the animals were OVX and euthanized at different times: 30 days (G30), 40 days (G40), 60 days (G60), and 80 days (G80). The animals in the experimental groups underwent bilateral ovariectomy to induce mineral loss. The femurs were collected after the periods established for each group and analyzed using microcomputed tomography (μCT) to determine bone density and count the number of trabeculae. Furthermore, the T-score was calculated for each group. Results: There were significant differences in bone density when comparing all groups, with GC > G30 > G40 > G60 > G80. For the number of trabeculae, GC presented more trabeculae than all other groups. More trabeculae were also observed in G30 when compared to G40, G60, and G80; however, there were no differences between G40, G60, and G80. Regarding the calculation of the T-score by group, osteopenia was observed in G30 (T-score: −2.42) and osteoporosis was observed in G40, G60, and G80 (T-scores: −4.38, −6.34, and −7.71, respectively). Conclusions: The results demonstrate that ovariectomy induces progressive changes in bone structure, with the onset of osteopenia 30 days after ovariectomy and osteoporosis after 40 days in this experimental model. These results may aid future investigations that seek to focus on the specific treatment of osteopenia and/or osteoporosis.

This study analyzed a recently developed bone substitute biomaterial made of chitosan-xanthan- hydroxyapatite-graphene oxide (CXHAG). The CXHAG particles underwent in vitro structural and morphological characterization, and in vivo testing with or without osteogenic conditioned medium from mesenchymal stem cells. Aim: The aim of this study was to determine whether the CXHAG novel biomaterial, supplemented with conditioned medium from mesenchymal stem cells, could be useful for bone regeneration. Materials and Method: For the in vitro study, cells were incubated with 20mg of CXHAG granules for 24 hours and a MTT assay was performed to tests for cytotoxicity. For the in vivo study, critical size calvarial bone defects were created in twenty-five rats. One animal had the defect unfilled (Control Group–CG) and was euthanized after 42 days. Twelve rats received the CXHAG particles (Group 1–G1) and the other twelve received the CXHAG particles supplemented with the conditioned medium (Group 2–G2). All G1/G2 grafts were covered with a CXHAG membrane. G1/G2 animals were euthanized after 14 days (T1) or 42 days (T2). The specimens were processed and histologically evaluated. Results: SEM analysis of the CXHAG particles showed granules of 300-400μm, with a rough irregular surface. They were not cytotoxic to dental pulp stem cells in vitro. The CG specimen showed loose immature connective tissue and no bone formation at the center of the defect. G1 and G2 presented remnant biomaterial particles at both time points, but only G2 had bone formation at the center of the defect. Conclusions: The conditioned medium had a positive effect on bone regeneration in rat calvarial critical size defects when associated with the novel bone substitute biomaterial.

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.

This study aimed to evaluate periosteum-derived mesenchymal stem cells (P-MSCs) cultured under simulated microgravity (SMG) conditions. P-MSCs were induced toward osteogenic differentiation and then exposed to SMG for up to 48 h. As a control, P-MSCs were maintained under identical conditions but without SMG exposure. Cell viability, osteogenesis-related analytes, and gene expression were analyzed at 3, 24 and 48 h. Cell viability under SMG was lower after 3 h but was significantly higher after 24 h, with no difference at 48 h. There was a higher expression of pathways associated with inflammation at 3 h, which was attenuated by 24 h and neutralized at 48 h. P-MSCs under SMG demonstrated three characteristics in at least one timepoint, which supports a pro-osteogenic signaling response: (1) higher osteoprotegerin levels; (2) lower DKK1 and TNF levels; (3) upregulation of genes related to osteogenesis. Our data suggest that P-MSCs exhibit enhanced pro-osteogenic regulatory modulation in SMG.

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.